JP7437385B2 - Poxvirus composition with improved storage - Google Patents

Description

FIELD OF THE INVENTION The present invention relates to the stabilization of poxvirus compositions for storage at refrigerator temperatures and/or −20° C. for longer periods, in particular for at least 12 months. The invention further relates to stabilized poxvirus compositions for storage at high temperatures and humidity. The invention also relates to methods of formulating such compositions for long-term storage.

BACKGROUND OF THE INVENTION Vaccinia viruses, particularly poxviruses including modified vaccinia Ankara (MVA) virus, have been used for vaccines against infectious diseases such as HIV, influenza, malaria, and respiratory syncytial virus (RSV), and for immunotherapy against cancer. and developed as a vector for oncolytic therapy (Choi and Chang Clin Exp Vaccine Res 2013, 2:97-105; Rezaee et al. Curr Opin Virol 2017, 24:70-78; Al Yaghchi et al. l.Immunotherapy 2015, 7: 1249-1258; Verheust et al. Vaccine 2012, 30: 2623-2632; Mastrangelo et al. J Clin Invest 2000, 105: 1031-1034). Several unique characteristics make poxviruses ideal candidates for vaccine development or gene delivery: (i) large packaging volume of recombinant DNA; (ii) regulated by a strong poxvirus promoter; (iii) loss of persistence or genomic integration in the host due to cytoplasmic replication; (iv) high immunogenicity as a vaccine; and (v) ease of manufacturing vectors and vaccines. (Verheust et al. Vaccine 2012, 30:2623-2632).

However, live attenuated vaccines pose challenges in formulation due to the complex macromolecular structure of the virus. This is even more difficult for large enveloped viruses such as poxviruses. For example, vaccinia viruses such as MVA consist of two very large envelopes (approximately 200-300 nm) with a size of approximately 192 kbps, consisting of a core region composed of viral DNA wrapped in a lipoprotein core membrane and various enzymes. It is a stranded DNA virus. The outer layer consists of a double lipid membrane envelope (Al Yaghchi et al. Immunotherapy 2015, 7: 1249-1258). There are two major morphologically distinct infectious types of virions: intracellular mature viruses (IMVs) and extracellular enveloped viruses (EEVs). IMV represents the majority of infectious particles that remain in the cytoplasm until cell lysis. EEVs are released from the cell and carry an extra lipid envelope containing at least 10 associated proteins not present in IMVs. This is an important consideration since the lipid membrane is very fragile and loss of the viral envelope will inactivate the virus. Stability can vary even more depending on the preparations and excipients used to prepare and store the purified virus.

One difficulty is storing water below the freezing point to avoid virus destabilization and/or destruction during freezing and thawing. To ensure stability, stocks of purified infectious viruses in the past were generally stored at temperatures below -60 degrees Celsius. One of the problems with storage at such low temperatures is the possibility of thawing and refreezing during transportation or at the site of administration.

The limits of stability of live viruses in aqueous compositions are well known, and most attenuated viruses are lyophilized products, such as the fully replication-competent vaccinia virus ACAM2000. ACAM2000 is a lyophilized product containing 6-8mM HEPES (pH 6.5-7.5), 2% human serum albumin, 0.5-0.7% sodium chloride, 5% mannitol, and trace amounts of neomycin and polymyxin B. Approved as a preparation. Lyophilized vaccinia virus was reconstituted in 50% (v/v) glycerol, 0.25% (v/v) phenol in water for injection (Berhanu et al. Vaccine 2010, 29:289-303).

Hekker et al. described a lyophilized smallpox vaccine composition containing a combination of peptone-sorbitol with 2% hemacel or 2% polyvinylpyrrolidone (Hekker et al. Journal of Biological Standardization 1973, 1:21-32, below) Abstract Burke et al. Crit Rev Ther Drug Carrier Syst 1999, 16:1-83). Further lyophilized compositions comprising MVA or ALVAC are described in WO03/053463, WO05/066333, WO07/056847, WO2011/121306, WO2014/053571, and Zhang et al. Chemical Research in Chinese Universities 2007, 23: 329-332. WO2010/135495 describes a method for stabilizing viruses in spray-dried powder compositions containing mannitol.

Just and Finke analyzed lyophilized MVA compositions containing stabilizers such as albumin, sorbitol, dextran, cysteine, hemacel, etc. and found that the lyophilized compositions had an excellent 5% (w/v) concentration when stored at +4°C. Sorbitol and 1% (w/v) human albumin were described. (Just and Finke Zentralbl Bakteriol Orig A1979, 245:276-282). They also analyzed the stability of non-lyophilized MVA suspensions containing 1% (w/v) human albumin, but found that these formulations showed a decrease in virus titer when stored at -70°C for more than 8 months. was observed.

Prabhu et al. (Prabhu et al. Biologicals 2014, 42:169-175) described three lyophilized camelpox vaccine formulations, which were prepared using 3.5% hydration in potassium phosphate buffer pH 6.2. Containing degraded gelatin and 3.5% sorbitol, it showed a reduction in virus titer even at 4°C after reconstitution.

Freeze-dried vaccines are typically more thermostable than non-freeze-dried alternatives, but freeze-drying involves cost, reconstitution before use, instability after reconstitution, and freeze and desiccation stress on virus particles. There are some drawbacks, such as (Capelle et al. Eur J Pharm Biopharm 2018, 129:215-221). Additionally, lyophilized vaccines are more prone to administration and dosing errors compared to liquid vaccines due to the need for reconstitution, which can lead to wasted vaccine or ineffective vaccine doses (Capelle et al. Eur J Pharm Biopharm 2018, 129:215-221).

There have also been attempts to use vaccinia virus for oral vaccine applications (US Pat. No. 6,969,345). The compositions described include mannitol with other ingredients such as hydroxyethyl starch, fish oil, glycerol, and gelatin.

Furthermore, liquid stabilization of live attenuated virus vaccines is the most difficult as the degradation kinetics and dynamic processes are more favorable (Tlaxca et al. Adv Drug Deliv Rev 2015, 93:56-78).

WO2010/056991 describes liquid or liquid freezing compositions comprising MVA virus and mannitol, where mannitol is the only stabilizer of the composition.

The beneficial effect on the stability of poxviruses using chelating agents and ethanol when stored for example at +5° C. for 12-24 months is disclosed in WO2016/087457.

WO2011/121301 describes the use of N,N-dimethylglycine or N,N,N-trimethylglycine for the stabilization of MVA in a liquid setting for one week at 37°C.

However, it allows for the stabilization of poxvirus-based materials, allowing for large-scale industrial applications, providing compositions for storage without affecting the biological activity of the product, and improving the desired properties of the virus. There remains a need for new formulations that make it possible to maintain and more specifically avoid or reduce loss of viral titer. Specifically, there is a need for a liquid pharmaceutical poxvirus preparation that provides long-term stability and does not require storage below -60°C. Additionally, it would be desirable to provide a high potency, small volume composition suitable for storage at refrigerator temperatures and/or temperatures of about -20°C.

Specifically, suitable for storage at about -20°C for about 1 year or more, followed by storage at +2 to +8°C (preferably for at least 6 or 9 months), and for subcutaneous, intramuscular and/or intranasal administration. There remains a need for the development of poxvirus liquid compositions that are compatible with. Liquid poxvirus compositions that are stable for about 1 year or more at +2 to +8°C are also desirable. Such liquid compositions offer advantages including low cost of goods, reduced development and/or manufacturing time, and user convenience. The present invention addresses these needs by disclosing improved poxvirus compositions, particularly MVA compositions that exhibit enhanced stability over extended periods of time at temperatures ranging from -20°C and/or +2 to +8°C. address and satisfy. Further disclosed are poxvirus compositions that exhibit enhanced stability at +25°C and 60% relative humidity.

The present invention provides aqueous compositions with improved stability of live poxviruses such as MVA. The inclusion of at least one disaccharide, sugar alcohol, gelatin, albumin, a pharmaceutically acceptable buffer, and at least one monovalent salt improves stability in both liquid and liquid frozen states.

Accordingly, in one aspect, the invention provides an aqueous composition comprising at least one poxvirus, a disaccharide, a sugar alcohol, gelatin, albumin, a pharmaceutically acceptable buffer, and at least one monovalent salt. , such compositions have a pH in the range of pH 7.0 to pH 8.5.

Another aspect of the invention provides a vaccine or pharmaceutical composition comprising an aqueous composition of the invention.

Another aspect of the invention provides a method of producing an aqueous live poxvirus composition, which method comprises the following steps:
a) providing a preparation comprising at least one poxvirus in a pharmaceutically acceptable buffer;
b) combining the poxvirus preparation of step a) with a solution comprising at least one disaccharide, a sugar alcohol, gelatin, albumin, a pharmaceutically acceptable buffer, and at least one monovalent salt; including;
Here, the above buffer solutions of a) and b) have a pH in the range of pH 7.0 to pH 8.5.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention.

Log ₁₀ (InfU/mL) potency titers of formulations (F17, F23, F9, F27) at -20°C compared to control formulation (10mM Tris, 140mM NaCl, pH 7.7). Average potency (infectivity) was determined by the fluorescence activated cell sorter (FACS) assay of Example 1. Log ₁₀ (InfU/mL) potency titers of formulations (F17, F23, F9, F27) at +5°C compared to control formulation (10mM Tris, 140mM NaCl, pH 7.7). Average potency (infectivity) was determined by the fluorescence-activated cell sorter (FACS) assay of Example 1. Viral titer, i.e. efficacy in log ₁₀ (InfU/mL) of formulations (F23, F55, F62, F66) at +25°C/60% relative humidity compared to control formulation (10mM Tris, 140mM, pH 7.7) titer. Average potency (infectivity) was determined by the fluorescence-activated cell sorter (FACS) assay of Example 1.

The long-term stability of the poxvirus, particularly MVA, present in the aqueous compositions of the present invention is due to the presence of at least one disaccharide, a sugar alcohol, gelatin, albumin, a pharmaceutically acceptable buffer. It has been found that the present invention is provided by a combination of a liquid and an excipient comprising at least one monovalent salt. Excellent protection could be obtained by those excipients included in aqueous compositions with a pH in the range of pH 7.0 to pH 8.5.

Accordingly, one aspect of the invention provides an aqueous composition comprising at least one poxvirus, at least one disaccharide, a sugar alcohol, gelatin, albumin, a pharmaceutically acceptable buffer, and at least one monovalent salt. and such compositions have a pH in the range of pH 7.0 to pH 8.5.

Poxviruses Poxviruses are generally enveloped viruses and are large viruses that carry double-stranded DNA. Poxviruses belong to the Poxviridae family and include 71 types of viruses classified into 16 genera (Virus Taxonomy: published in 2017). Two of the best known orthopoxviruses are the smallpox virus, which is the causative agent of smallpox, and the vaccinia virus, whose conversion into a vaccine made it possible to eradicate smallpox.

Poxviruses such as vaccinia virus are known to those skilled in the art and have been used to generate recombinant vaccines in the fight against infectious organisms and more recently cancer (Mastrangelo et al. J Clin Invest 2000, 105: 1031-1034).

Within the context of this disclosure, poxviruses preferably include orthopoxviruses or avipoxviruses. In a preferred embodiment of the invention, the poxvirus is an orthopoxvirus.

Orthopoxviruses include, but are not limited to, smallpox virus, vaccinia virus, cowpox virus, and monkeypox virus. Preferably, the orthopoxvirus is vaccinia virus.

The term "vaccinia virus" is used, for example, in Ankara, VACV Western Reserve (WR), VACV Copenhagen (VACV-COP), Temple of Heaven, Paris, Budapest, Dairen, Gam. , MRIVP, Per, Tashkent, TBK, Tian Tan, Tom, Bern, Patwadangar, BIEM, B-15, EM-63, IHD-J, IHD-W, Ikeda, DryVax (VACV Wyeth or New York City Health Commissioner [NYCBH] strain), NYVAC, ACAM1000, ACAM2000, Vaccinia Lister (also known as Elstree), LC16mO or LC16m8.

In a further embodiment, the poxvirus of the invention is an MVA virus.

MVA virus was generated by 516 serial passages of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblast cells (for a review, see Mayr et al. Infection 1975, 3:6-14). ). As a result of these long-term passages, the resulting MVA virus genome was described as an advanced host cell with restricted replication into avian cells, as approximately 31 kilobases of its genomic sequence was deleted (Meyer et al. J Gen Virol 1991, 72 (Pt 5): 1031-1038). In various animal models, the resulting MVA was shown to be significantly non-toxic compared to fully replicable starting material (Mayr and Danner Dev Biol Stand 1978, 41:225-234 ).

MVA viruses useful in the practice of the present invention include, but are not limited to, MVA-572 (deposited as ECACC V94012707 on January 27, 1994); MVA-575 (deposited as ECACC V94012707 on December 7, 2000); (deposited as ECACC V00120707), MVA-I721 (cited in Suter et al. Vaccine 2009, 27:7442-7450), NIH clone 1 (deposited as ATCC® PTA-5095 on March 27, 2003), and MVA-BN (deposited with the European Cell Culture Collection (ECACC) on August 30, 2000 under number V00083008) may be mentioned.

More preferably, the MVA used according to the invention includes MVA-BN and MVA-BN derivatives. MVA-BN is described in International PCT Publication WO 02/042480. "MVA-BN derivative" means any virus that exhibits essentially the same replication characteristics as MVA-BN, but exhibits differences in one or more portions of their genome, as described herein. Point.

MVA-BN and MVA-BN derivatives are replication incompetent, meaning that they fail to replicate in vivo and in vitro. More specifically, in vitro, MVA-BN or MVA-BN derivatives are reproductively replicable in chicken embryo fibroblasts (CEF), but not in the human keratinocyte cell line HaCat (Boukamp et al (1988), J. Cell Biol. 106:761-771), human osteosarcoma cell line 143B (ECACC deposit no. 91112502), human embryonic kidney cell line 293 (ECACC deposit no. 85120602), and human cervical adenocarcinoma cell line HeLa (ATCC deposit no. CCL -2) states that it is incapable of reproduction. Furthermore, MVA-BN or MVA-BN derivatives have a viral amplification ratio that is at least 2-fold, more preferably 3-fold lower than MVA-575 in Hela cells and HaCaT cell lines. Tests and assays for these properties of MVA-BN and MVA-BN derivatives are described in WO 02/42480 (US Patent Application No. 2003/0206926) and WO 03/048184 (US Patent Application No. 2006/0159699). .

As explained in the previous paragraph, the term "reproductively incompetent" or "reproductively incompetent" in human cell lines in vitro is described, for example, in WO 02/42480, which also Methods for obtaining MVAs having desirable properties such as those described above are also taught. This term applies to viruses that have a viral amplification factor of less than 1 in vitro at 4 days after infection using the assays described in WO 02/42480 or US Pat. No. 6,761,893.

The term "failure of reproductive replication" refers to a virus that has a viral amplification rate in human cell lines in vitro, as described in the previous paragraph, of less than 1 at 4 days post infection. The assays described in WO 02/42480 or US Pat. No. 6,761,893 are applicable for determining the rate of viral amplification.

As explained in the previous paragraph, the amplification or replication of viruses in human cell lines in vitro is commonly referred to as the "amplification factor," which is the difference between the amount of virus produced from the infected cell (the output) and the amount of virus that enters the cell in its initial location. It is expressed as a ratio of the amount originally used to infect (input). An amplification factor of "1" defines an amplification state in which the amount of virus produced from an infected cell is the same as the amount originally used to infect the cell, i.e., an infected cell is defined as a This means that playback is allowed. In contrast, an amplification factor of less than 1, ie, a decrease in output compared to the input level, indicates a lack of reproductive replication and thus attenuation of the virus.

In another embodiment, the poxvirus of the invention is an avipoxvirus, such as, but not limited to, fowlpox virus.

The term "avipoxvirus" includes fowlpox virus, canarypox virus, Uncopoxvirus, Mynahpoxvirus, pigeonpox virus, Psittacinepoxvirus, quailpox virus, peacockpox virus, Refers to any avipox virus, such as penguinpox virus, sparrowpox virus, starlingpox virus, and turkeypox virus. Preferred avipox viruses are canarypox virus and fowlpox virus.

Avipoxvirus is a genus of the Poxviridae family, and the virus can infect and replicate in birds, but not in non-avian species (Vanderplasschen and Pastoret Curr Gene Ther 2003, 3:583-595). Avipoxviruses, such as fowlpox virus, have been shown to be safe and effective non-replicating vectors when used in non-avian species. (Ibid.).

An example of a canarypox virus is the Rentschler strain. A plaque-purified canarypox strain called ALVAC (US Pat. No. 5,766,598) has been deposited with the American Type Culture Collection (ATCC) under the terms of accession number VR-2547 and the Budapest Treaty. Another canarypox strain is available from Institute Merieux, Inc. There is a commercially available canarypox vaccine strain called LF2 CEP 524 24 10 75 available from .

Examples of fowlpox viruses are FP-1 strain, FP-5 strain, TROVAC (U.S. Pat. No. 5,766,598), POXVAC-TC (U.S. Pat. No. 7,410,644), TBC-FPV (Therion Biologics-FPV). FP-1 is a Duvette strain modified for use as a vaccine in one-day-old chickens. This strain is a commercially available fowlpox virus vaccine strain designated O DCEP 25/CEP67/2309 October 1980 and is a commercial fowlpox virus vaccine strain designated by the Institute Merieux, Inc. Available from. FP-5 was manufactured by American Scientific Laboratories (Division of Schering Corp.) Madison, Wis. , United States Veterinary License No. 165, serial No. 30321 is a commercially available fowlpox virus vaccine strain derived from chickenpox.

In another embodiment, the poxvirus of any embodiment of the invention or any preferred poxvirus is a live virus.

The poxvirus (especially orthopoxvirus, more specifically vaccinia virus or preferably MVA) preferably has a concentration of at least 10 ⁷ InfU/mL, preferably at least 2×10 ⁷ InfU/mL, at least 3×10 ⁷ InfU/mL. mL, at least 5 x ¹⁰ InfU/mL, at least 6 x ¹⁰ InfU/mL, at least 7 x ¹⁰ InfU/mL, at least 8 x ¹⁰ InfU/mL, at least 9 x ¹⁰ InfU/mL, at least 1x 10 ⁸ InfU/mL, at least 2 x 10 ⁸ InfU/mL, at least 3 x 10 ⁸ InfU/mL, at least 4 x 10 ⁸ InfU/mL, at least 5 x 10 ⁸ InfU/mL, at least 6 x 10 ⁸ InfU/mL , at least 7×10 ⁸ InfU/mL, at least 8×10 ⁸ InfU/mL, at least 9×10 ⁸ InfU/mL, at least 1×10 ⁹ InfU/mL, at least 2×10 ^{9 Inf} U/mL, at least 3×10 ^{The aqueous composition or _} present in the method of the invention. For practical reasons, poxviruses (particularly orthopoxviruses, more specifically vaccinia viruses or preferably MVA) may contain up to 1 x 10 ¹¹ InfU/mL, up to 5 x 10 ¹⁰ InfU/mL, or preferably A titer of up to 1×10 ¹⁰ InfU/mL is present in the aqueous composition or method of the invention.

Specifically, poxviruses (particularly orthopoxviruses, more specifically vaccinia virus or preferably MVA) can be used in the present invention at a titer of about 1×10 ⁷ InfU/mL to 1×10 ¹¹ InfU/mL. present in an aqueous composition or method.

Specifically, poxviruses (particularly orthopoxviruses, more specifically vaccinia virus or preferably MVA) can be purified according to the invention at a titer of about 1×10 ⁷ InfU/mL to 5×10 ¹⁰ InfU/mL. present in an aqueous composition or method.

Specifically, poxviruses (particularly orthopoxviruses, more specifically vaccinia virus or preferably MVA) can be purified according to the invention at a titer of about 1×10 ⁷ InfU/mL to 1×10 ¹⁰ InfU/mL. present in an aqueous composition or method.

Specifically, poxviruses (particularly orthopoxviruses, more specifically vaccinia virus or preferably MVA) can be used in the present invention at a titer of about 1 x 10 ⁷ InfU/mL to 6 x 10 ⁹ InfU/mL. present in an aqueous composition or method.

In certain embodiments, the aqueous compositions provided herein are administered in a single dose or in multiple (i.e., 2, 3, 4, etc.) doses, preferably between 0.1 and 0.5 ml. Administered to the subject by volume. In certain embodiments, the aqueous compositions provided herein are administered to a subject at a dose of 10 ⁷ to 10 ¹⁰ InfU of virus in the aqueous composition, preferably in a volume of 0.1 to 0.5 ml. be done. In certain other embodiments, the aqueous composition is administered in a first (priming) inoculation and one or more subsequent booster doses. In a particular embodiment, the first dose comprises 10 ⁷ to 10 ¹⁰ InfU of poxvirus in an aqueous composition, and the second dose comprises 10 7 InfU, preferably in a volume ^of 0.1 to 0.5 ml. Contains an aqueous composition of ~10 ¹⁰ InfU of virus.

In certain embodiments, the one or more subsequent booster doses include the same recombinant poxvirus as previously administered, and the method includes a homologous prime-boost vaccination. including. In certain embodiments, the one or more subsequent booster doses comprises a different recombinant poxvirus than previously administered, and the method comprises a homologous prime-boost vaccination. including.

In certain embodiments, one or more subsequent administrations (i.e., one or more booster vaccinations) are administered at intervals that include days, weeks, or months after the administration of the initial priming vaccination. be done. In certain embodiments, one or more subsequent administrations of recombinant poxvirus (i.e., one or more booster vaccinations) are administered one or more times after the administration of an initial dose of recombinant poxvirus (i.e., priming vaccination). Administered at intervals of 2, 3, 4, 5, 6, 7 or more weeks. In certain embodiments, one or more subsequent administrations of recombinant poxvirus (i.e., one or more booster vaccinations) are administered 1, 2, 3, 4, 5, Administered at intervals of 6, 7, 8, 9, 10, 11, 12 or more months.

The poxvirus (particularly orthopoxvirus, more particularly vaccinia virus or preferably MVA) included in the compositions or methods of the invention may be a wild-type poxvirus, an attenuated poxvirus or a recombinant poxvirus.

The term "recombinant" virus in any embodiment described herein refers to a virus that contains exogenous nucleic acid sequences inserted into its genome that are not naturally present in the parent virus, more specifically poxviruses. refers to Therefore, a recombinant virus (e.g., specifically an orthopoxvirus, more specifically a vaccinia virus or preferably an MVA) is a virus that does not exist in nature or that is different in an arrangement that is not found in nature. Refers to a virus created by the artificial combination of two or more segments of nucleic acid sequences of synthetic or semi-synthetic origin linked to a nucleic acid. Artificial combinations are most commonly accomplished by artificially manipulating isolated segments of nucleic acids using well-established genetic engineering techniques. In general, "recombinant" poxviruses as described herein refer to poxviruses produced by standard genetic engineering methods; thus, for example, the MVA viruses of the invention are genetically engineered or genetically modified. This is the MVA virus. Accordingly, the term "recombinant MVA" includes an MVA virus (eg, MVA-BN) that has integrated into its genome at least one recombinant nucleic acid, preferably in the form of a transcription unit. A transcription unit may include a promoter, enhancer, terminator and/or silencer. Recombinant MVA viruses of the invention are capable of expressing heterologous antigenic determinants, polypeptides or proteins (antigens) upon induction of regulatory elements, such as promoters.

Methods for producing recombinant poxviruses Methods for obtaining recombinant poxviruses (e.g., VACV or MVA) or for inserting exogenous coding sequences into the poxvirus (e.g., VACV or MVA) genome are well known to those skilled in the art. be. For example, methods for standard molecular biology techniques such as DNA cloning, DNA and RNA isolation, Western blot analysis, RT-PCR and PCR amplification techniques are described in Molecular Cloning, Laboratory Manual 2. ^nd Ed. (J. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)) and techniques for handling and manipulating viruses are described in the Virology Methods Manual (B.W.J. Mahy et al. (eds. ), Academic Press (1996)). Similarly, techniques and know-how for handling, manipulating, and genetically engineering poxviruses are described in Molecular Virology: A Practical Approach (A. J. Davison & R. M. Elliott (Eds.), The Practical Approach Series, IRL Press at Oxford University Press, Oxford, UK (1993), for example, Chapter 9: Expression of genes by Vaccinia virus vectors); Current Protocols i n Molecular Biology (John Wiley & Son, Inc. (1998), e.g., Chapter 16, Section IV: Expression of proteins in mammalian cells using vaccinia viral vector); and Genetic Engineering, Recent Developo pments in Applications, Apple Academic Press (2011), Dana M. Santos, for example, Chapter 3: Recombinant-mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA)). Construction and isolation of recombinant MVA is also described in Methods and Protocols, Vaccinia Virus and Poxvirology, ISBN 978-1-58829-229-2 (Staib et al.), Humana Press (2004). For example, See also Chapter 7.

Methods for producing large quantities of recombinant poxviruses and purifying viral-based materials such as viral vectors and/or viruses used in accordance with the present invention are known to those skilled in the art. Available methods include CEF cells or cell lines, specifically DF-1 (US Pat. No. 5,879,924), EBx chicken cell line (WO2005/007840), EB66 duck cells (WO08/129058), or Cairina moschata, including replication of the virus in immortalized avian cells (WO2007/077256 or WO2009/004016). They can be cultured under conditions well known to those skilled in the art. Serum-free methods are preferred for virus culture and virus amplification. Certain serum-free methods for virus culture and virus amplification in CEF cells are described, for example, in WO2004/022729. Upstream and downstream processes for producing viruses are well known to those skilled in the art. They are available from WO2012/010280 or WO2016/087457. Methods useful for purifying the viruses of the present application are disclosed in WO03/054175, WO07/147528, WO2008/138533, WO2009/100521 and WO2010/130753. Exemplary methods for propagation and purification of recombinant poxviruses in duck embryo-derived cells are described by Leon et al. Vaccine 2016, 34:5878-5885.

Exemplary Production of Recombinant MVA Viruses Different methods may be applicable for production of the various recombinant MVA viruses disclosed herein. The DNA sequence inserted into the virus is an E. coli in which DNA homologous to a section of poxvirus DNA has been inserted. E. coli plasmid constructs. Alternatively, the DNA sequence to be inserted may be ligated to the promoter. The promoter-gene linkage may be placed in the plasmid construct such that the promoter-gene linkage is flanked on both ends by DNA homologous to DNA sequences that flank the region of poxvirus DNA containing the non-essential locus. The resulting plasmid construct was constructed using E. They may be amplified by growth in E. coli bacteria or isolated. The isolated plasmid containing the DNA gene sequence to be inserted may be transfected into a cell culture of, for example, chicken embryo fibroblasts (CEF) at the same time as the culture is infected with MVA virus. Recombination between homologous MVA viral DNA and the viral genome within the plasmid can generate poxviruses modified by the presence of foreign DNA sequences, respectively.

According to a preferred embodiment, cells in a suitable cell culture, eg CEF cells, may be infected with the MVA virus. The infected cells may then be transfected with a first plasmid vector containing foreign or heterologous gene(s), such as one or more nucleic acids provided in this disclosure (preferably a poxvirus under the transcriptional control of expression control elements). As explained above, plasmid vectors also contain sequences that can direct the insertion of exogenous sequences into selected portions of the MVA viral genome. Optionally, the plasmid vector also includes a cassette containing a marker and/or selection gene operably linked to a poxvirus promoter. The use of selection or marker cassettes simplifies the identification and isolation of the recombinant poxviruses produced. However, recombinant poxviruses can also be identified by PCR techniques. Subsequently, additional cells may be infected with the recombinant poxvirus obtained as described above and may be transfected with a second vector containing a second foreign or heterologous gene(s). . If this gene is introduced into a different insertion site in the poxvirus genome, this second vector also contains poxvirus homologous sequences that direct the integration of the second foreign gene(s) into the poxvirus genome. different. After homologous recombination has occurred, a recombinant virus containing two or more foreign or heterologous genes may be isolated. In order to introduce additional foreign genes into the recombinant virus, the infection and transfection steps can be performed by using the recombinant virus isolated in the previous step for infection and a further step for transfection. It may be repeated by using additional vectors containing foreign gene(s).

In other embodiments, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment of the invention comprises a nucleic acid encoding an antigen, preferably at least one antigen. including.

For example, suitable antigens according to the invention include one or more introduced molecules having an open reading frame encoding one or more polypeptide(s) against which an immune response is desired when the virus is used for vaccination purposes. May include gene(s). Examples include, but are not limited to, immune responses to viruses or pathogens, including, but not limited to, RSV, HIV, HPV, HBV, malaria, Ebola, MARV, FMDV, dengue, equine encephalitis virus, or any combination thereof. One or more transgenes suitable for production.

In preferred embodiments, the antigen is a viral antigen, costimulatory molecule, and/or tumor-associated antigen (TAA).

In a preferred embodiment of the invention, the viral antigen is a filovirus, a picornavirus, a papillomavirus, a hepatitis virus, a flavivirus, a retrovirus, an orthomyxovirus, an equine encephalitis virus, a paramyxovirus, and/or a combination thereof. An immunogenic antigen selected from

In a preferred embodiment of the invention, the immunogenic antigen is a protein, preferably a full-length protein.

In a preferred embodiment of the invention, the paramyxovirus is respiratory syncytial virus (RSV), for example as described in WO2014/019718.

In another preferred embodiment, the recombinant poxvirus (especially orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment of the invention comprises a nucleic acid encoding an RSV antigen.

In another preferred embodiment, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment comprises a nucleic acid encoding an RSV protein.

In another preferred embodiment, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment comprises a nucleic acid encoding RSV F glycoprotein.

In another preferred embodiment, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment comprises a nucleic acid encoding RSV F glycoprotein and RSV G glycoprotein. include.

In another preferred embodiment, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment encodes an RSV F glycoprotein and two RSV G glycoproteins. Contains nucleic acids.

In another preferred embodiment, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment comprises RSV F glycoprotein, two RSV G glycoproteins and RSV N Contains nucleic acids that encode proteins.

In another preferred embodiment, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment comprises an RSV F glycoprotein, two RSV G glycoproteins, an RSV N Contains a protein and a nucleic acid encoding an RSV matrix protein.

In another preferred embodiment, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment comprises an RSV F glycoprotein, two RSV G glycoproteins, an RSV N protein and a nucleic acid encoding the RSV M2-1 protein.

In a preferred embodiment of the invention, the filovirus is Ebola virus and/or Marburg virus (MARV), for example as described in WO2016/036955, WO2016/036971 or WO2016/034678.

In a preferred embodiment of the invention, the picornavirus is foot and mouth disease virus (FMDV), for example as described in WO2016/202828.

In a preferred embodiment of the invention, the papillomavirus is a human papillomavirus, for example as described in WO2017/192418, WO90/10459, WO05/09241, WO98/04705, WO99/03885 or WO2007/121894.

In a preferred embodiment of the invention, the hepatitis virus is selected from the group of hepatitis A virus, hepatitis B virus, hepatitis C virus and hepatitis E virus, for example as described in WO2004/111082.

In a preferred embodiment of the invention, the flavivirus is Dengue virus (DENV).

In a preferred embodiment of the invention, the retrovirus is HIV-1.

In a preferred embodiment of the invention, the orthomyxovirus is an influenza virus.

In a preferred embodiment of the invention, the equine encephalitis virus (EEV) is, for example, Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV) and/or Venezuelan equine encephalitis virus, as described in WO2017/129765. It is a virus (VEEV).

In a preferred embodiment of the invention, the viral antigen is immunogenic selected from the group of RSV, Ebola virus, MARV, FMDV, HPV, HBV, HIV, influenza virus, DENV, RSV, EEV and any combination thereof. It is an antigen.

A variety of costimulatory molecules are known to those skilled in the art. These include, but are not limited to, ICAM-1, LFA-3, CD72, B7-1, B7-2, CD40, CD40 ligand (CD40L) or other B7-related molecules, or combinations thereof, such as TRICOM. can be mentioned.

The "TRICOM." triad of costimulatory molecules (also known as TRICOM) includes B7-1 (also known as B7.1 or CD80), intracellular adhesion molecule-1 (ICAM-1), Lymphocyte function-associated antigen-3 (LFA-3, also known as CD54) and lymphocyte function-associated antigen-3 (LFA-3, also known as CD58), and generally target specific antigens to increase antigen-specific immune responses. contained in a recombinant viral vector (e.g., a poxvirus vector) that expresses The individual components of TRICOM may be under the control of the same or different promoter(s) and may be provided on the same vector as the particular antigen or on separate vectors. Exemplary vectors are described, for example, by Hodge et al. Cancer Res 1999, 59:5800-5807 et al. , “A Triad of Costimulatory Molecules Synergize to Amplify T-Cell Activation,” Cancer Res. 59:5800-5807 (1999) and US Pat. No. 7,211,432B2, both of which are incorporated herein by reference.

TAA is well known to those skilled in the art and refers to self-cell antigens that are detected at higher frequency or density in tumor tissues or cells compared to non-tumor tissues or cells.

In a preferred embodiment, the TAA is CEA, MUC-1, TRP-1, NY-ESO-1, TRP-2, p53, PSA, HER-2, PAP, Survivin, TYRP1, TYRP2, or Brachyury. selected from the group or any combination thereof.

In other embodiments, the recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any embodiment of the invention comprises a nucleic acid encoding a combination of TAAs. Such exemplary combinations may include HER2 and Brachyury, CEA and MUC-1, or PAP and PSA.

Stabilizers of Compositions The disaccharide according to the invention can be trehalose, sucrose, or a combination thereof. In a preferred embodiment of the invention, the disaccharide is trehalose or sucrose.

In other embodiments, the aqueous compositions or methods according to the invention contain between 2% (w/v) and 12% (w/v), preferably between 4% (w/v) and 12% (w/v). Contains a disaccharide (preferably trehalose or sucrose) in a range of concentrations. Specifically, the aqueous composition or method according to the invention comprises a concentration of a disaccharide (preferably trehalose or sucrose), which concentration ranges from 2% (w/v) to 11% (w/v); 2% (w/v) ~ 10% (w/v), 2% (w/v) ~ 9% (w/v), 2% (w/v) ~ 8% (w/v), 2% (w/v) ~ 7% (w/v), 2% (w/v) ~ 6% (w/v), 2% (w/v) ~ 5% (w/v), 4% (w /v) ~ 12% (w/v), 4% (w/v) ~ 11% (w/v), 4% (w/v) ~ 10% (w/v), 4% (w/v) ) ~9% (w/v), 4% (w/v) ~8% (w/v), 4% (w/v) ~7% (w/v), 5% (w/v) ~ 12% (w/v), 5% (w/v) to 11% (w/v), 5% (w/v) to 10% (w/v), 5% (w/v) to 9% (w/v), 5% (w/v) to 8% (w/v), 6% (w/v) to 12% (w/v), 6% (w/v) to 11% (w /v), 6% (w/v) to 10% (w/v), 6% (w/v) to 9% (w/v), 6% (w/v) to 8% (w/v) ), 7% (w/v) to 12% (w/v), 7% (w/v) to 11% (w/v), 7% (w/v) to 10% (w/v), It ranges from 7% (w/v) to 9% (w/v), or from 7% (w/v) to 8% (w/v).

In other embodiments, an aqueous composition or method according to the invention comprises a disaccharide at a concentration of 10% (w/v).

In other embodiments, an aqueous composition or method according to the invention comprises trehalose at a concentration ranging from 4% (w/v) to 12% (w/v).

In other embodiments, an aqueous composition or method according to the invention comprises trehalose at a concentration of 10% (w/v).

In other embodiments, an aqueous composition or method according to the invention comprises sucrose at a concentration ranging from 4% (w/v) to 12% (w/v).

In other embodiments, an aqueous composition or method according to the invention comprises sucrose at a concentration of 10% (w/v).

In other embodiments, aqueous compositions or methods according to the invention include sorbitol.

In other embodiments, aqueous compositions or methods according to the invention include sorbitol at a concentration ranging from 0.2% (w/v) to 5% (w/v).

In other embodiments, an aqueous composition or method according to the invention comprises sorbitol at a concentration ranging from 0.2% (w/v) to 4% (w/v).

In other embodiments, the aqueous compositions or methods according to the invention are comprised of 0.5% (w/v) to 4% (w/v), preferably 0.5% (w/v) to 3% (w/v) w/v).

In other embodiments, the aqueous compositions or methods according to the invention have a concentration in the range of 0.2% (w/v) to 2.2% (w/v), preferably 0.5% (w/v). v) and sorbitol at concentrations ranging from 2.2% (w/v).

In other embodiments, the aqueous compositions or methods according to the invention are provided at concentrations ranging from 1% (w/v) to 4% (w/v), preferably from 1% (w/v) to 3% (w/v). w/v).

In other embodiments, an aqueous composition or method according to the invention comprises sorbitol at a concentration of 2% (w/v).

In other embodiments, an aqueous composition or method according to the invention comprises sorbitol at a concentration of 2% (w/v).

Gelatin used in the present invention is well known to those skilled in the art. Gelatin is a naturally occurring water-soluble protein, either gelled or non-gelled, that is found in the bones, hides, and skin, tendons, and sinew of animals such as pigs, cows, fish, and poultry. can be obtained by partial hydrolysis of collagen produced from. Type A gelatin is produced by acid treatment of collagen raw materials, while B type gelatin is produced by alkali treatment of collagen raw materials. Gelatins for pharmaceutical formulations may include, for example, purified gelatins as described in the European Pharmacopoeia (Ph. Eur.) or the United States Pharmacopeia (USP).

The term "gelatin hydrolyzate" according to the present invention is also referred to as hydrolyzed gelatin, hydrolyzed collagen, collagen hydrolyzate, collagen peptide, gelatin hydrolyzate and hydrolyzed gelatin. These terms may be used interchangeably.

The term "gelatin hydrolyzate" means either a hydrolyzed polypeptide obtained by degrading gelatin by hydrolytic cleavage, or a polypeptide obtained by polymerizing the hydrolyzed polypeptides described above. The gelatin hydrolyzate is water soluble and preferably has a molecular weight of about 35,000 or less. Examples of gelatins that can be used in the present invention include VacciPro® (trade name for hydrolyzed gelatin or its chemical derivatives manufactured and sold by Gelita®, AG, Germany), Gelysate® (trade name for hydrolyzed gelatin or its chemical derivatives manufactured and sold by BBL Co., Ltd., USA), and Rousselot® Pharmaceutical Gelatin (manufactured and sold by Rousselot B.V., NL). Examples include commercially available products such as hydrolyzed gelatin or its chemical derivatives (trade names).

In other embodiments, the gelatin of the aqueous compositions or methods of the invention is preferably bovine or porcine gelatin, preferably porcine or bovine gelatin hydrolysate. Porcine gelatin is preferably used.

In other embodiments of the invention, the gelatin is porcine A-type gelatin. In other embodiments of the invention, the gelatin is porcine B-type gelatin.

In other embodiments, the aqueous compositions or methods of the invention contain gelatin or gelatin hydrolysate, or any preferred concentration of between about 0.02% (w/v) and 5% (w/v). Contains gelatin. In further embodiments, the aqueous compositions or methods of the invention comprise gelatin or gelatin hydrolysate, or any preferred gelatin at a concentration of about 0.1% (w/v) to 5% (w/v). . In other embodiments, the aqueous compositions or methods of the invention contain gelatin or gelatin hydrolysate, or any preferred gelatin at a concentration of about 0.2% (w/v) to 5% (w/v). include. In other embodiments, the aqueous compositions or methods of the invention contain gelatin or gelatin hydrolysate, or any preferred gelatin at a concentration of about 0.25% (w/v) to 5% (w/v). include. In other embodiments, the aqueous compositions or methods of the invention contain gelatin or gelatin hydrolysate, or any preferred gelatin at a concentration of about 0.25% (w/v) to 4% (w/v). include. In other embodiments, the aqueous compositions or methods of the invention contain gelatin or gelatin hydrolysate, or any preferred gelatin at a concentration of about 0.2% (w/v) to 3.2% (w/v). Contains gelatin. In other embodiments, the aqueous compositions or methods of the invention contain gelatin or gelatin hydrolysate, or about 1% (w/v) to 3.2% (w/v), preferably 2.5% (w/v) (w/v) to 3% (w/v) of any preferred gelatin. In other embodiments, the aqueous compositions or methods of the invention contain gelatin or gelatin hydrolysate, or any preferred gelatin at a concentration of about 0.2% (w/v) to 1.2% (w/v). Contains gelatin. In other embodiments, the aqueous compositions or methods of the invention contain gelatin or gelatin hydrolysate, or about 0.25% (w/v), 0.5% (w/v), 1% (w/v) v), 2% (w/v) or 3% (w/v) of any preferred gelatin.

In other embodiments, aqueous compositions or methods according to the invention include albumin. Albumin is a protein naturally found in mammalian plasma and is the most abundant protein. It also plays an important role in maintaining the desired osmotic pressure of the blood and in the transport of various substances in the bloodstream. The albumin used in the present invention is preferably human, pig, mouse, rat, rabbit, or goat albumin, but WO2011/051489, WO2011/051489, WO2010/092135, WO2012/150319, WO2014/072481, Described in WO2011124718, WO 2015/036579, WO2018/065491, WO2017/029407, WO2013/075066, or Otagiri and Chuang Biol Pharm Bull 2009, 32:527-534 It also includes variants such as Those skilled in the art will also recognize that modifications can be made to albumin by any means known in the art, such as recombinant DNA technology, post-translational modification, proteolytic cleavage, and/or chemical means. Will. Those substitutions and modifications to albumin that provide essentially equivalent stabilizing function to albumin without the substitution or modification are contemplated herein. Preferably the albumin is human serum albumin. In other embodiments, the albumin is recombinant albumin, preferably recombinant human serum albumin expressed and purified from Pichia pastoris, Saccharomyces cerevisiae or Oryza sativa. Preferably, the albumin used according to the invention is Recombumin® expressed in Saccharomyces cerevisiae. However, other recombinant albumins are suitable for the present invention, such as human recombinant albumin expressed in Pichia pastoris (e.g. Albagen™, rHSA, CAS No. 70024-90-7, Sigma- Aldrich).

In other embodiments, the aqueous composition or method according to the invention is provided at a concentration ranging from 0.02% (w/v) to 3% (w/v), preferably 0.02% (w/v). Albumin (preferably any preferred albumin mentioned herein) at a concentration ranging from 2% (w/v).

In other embodiments, the aqueous composition or method according to the invention is provided at a concentration in the range of 0.2% (w/v) to 2% (w/v), preferably 0.2% (w/v). Contains albumin (preferably any preferred albumin mentioned herein) at a concentration ranging from 1.5% (w/v).

In other embodiments, the aqueous composition or method according to the invention is provided at a concentration in the range of 0.5% (w/v) to 2% (w/v), preferably 0.5% (w/v). Contains albumin (preferably any preferred albumin mentioned herein) at a concentration ranging from 1.5% (w/v).

In other embodiments, the aqueous compositions or methods according to the invention contain albumin (preferably as referred to herein) at a concentration ranging from 0.8% (w/v) to 1.2% (w/v). any preferred albumin).

In other embodiments, the aqueous composition or method according to the invention comprises albumin (preferably any preferred albumin mentioned herein) at a concentration of 1% (w/v).

Buffer and pH
Aqueous compositions or methods according to any embodiment of the invention preferably have a pH in the range of pH 7.0 to pH 8.5.

In other embodiments of the invention, an aqueous composition or method according to any embodiment has a pH in the range of pH 7.3 to pH 8.1.

In other embodiments of the invention, an aqueous composition or method according to any embodiment has a pH of 7.7.

Those skilled in the art of drug development are well aware of buffers that can be used to achieve a pH of, for example, pH 7.0 to pH 8.5. Such buffers are preferably phosphate buffer, Tris (tris(hydroxymethyl)aminomethane), Tris-HCl (tris(hydroxymethyl)aminomethane-HCl), Trisine (N-[tris(hydroxymethyl)aminomethane-HCl), ) methyl)-methyl]-glycine), and HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid). The phosphate buffer preferably comprises a mixture of Na ₂ HPO ₄ and KH ₂ PO ₄ or a mixture of Na ₂ HPO ₄ and NaH ₂ PO ₄ . In certain embodiments, the buffer of the aqueous composition is a Tris buffer, preferably a Tris-HCl buffer.

In certain embodiments, an aqueous composition or method according to any embodiment does not include citrate or citrate buffer.

In certain embodiments, the buffer of an aqueous composition or method according to any embodiment is preferably present at a concentration in the range of 1 mM to 50 mM, preferably in the range of 1 mM to 25 mM.

In certain embodiments, the buffer of an aqueous composition or method according to any embodiment is present at a concentration in the range of 1mM to 15mM, preferably in the range of 5mM to 11mM, more preferably in the range of 7mM to 10mM.

In certain embodiments, the buffer of an aqueous composition or method according to any embodiment is present at a concentration of 10 mM.

In certain embodiments, the buffer of an aqueous composition or method according to any embodiment is present at a concentration of 7.5 mM.

Salts In other embodiments, an aqueous composition or method according to any embodiment includes a monovalent salt. The monovalent salt is preferably sodium chloride (NaCl) or potassium chloride (KCl), preferably NaCl. The NaCl is preferably present in a concentration between 40mM and 200mM, preferably between 40mM and 150mM.

In another embodiment, NACL is 40mm to 140mm, 40mm to 130mm, 40mm to 120mm, 40mm to 110mm, 40mm to 100mm, 40 mm to 90mm, 40 mm to 80mm, 50 mm ~ 150mm, 50 mm, 50 mm. 50mm to 130mm, 50mm ~120mM, 50mM-110mM, 50mM-100mM, 50mM-90mM, 50mM-80mM, 60mM-150mM, 60mM-140mM, 60mM-130mM, 60mM-120mM, 60mM-110mM, 60mM-100mM, 60mM-90mM , 60mM to 80mM , 70mM to 150mM, 70mM to 140mM, 70mM to 130mM, 70mM to 120mM, 70mM to 110mM, 70mM to 100mM, 70mM to 90mM, or 70mM to 80mM.

In another embodiment, NaCl may be present at a concentration of 60mM to 80mM.

In another embodiment, NaCl may be present at a concentration of 70mM.

In other embodiments, an aqueous composition or method according to any embodiment includes a divalent salt. The divalent salt is preferably magnesium chloride (MgCl ₂ ). The MgCl ₂ is preferably present in a concentration between 1mM and 300mM.

In another embodiment, _MgCl2 is 2mM to 300mM, 5mM to 300mM, 10mM to 300mM, 20mM to 300mM, 30mM to 300mM, 40mM to 300mM, 50mM to 300mM, 60mM to 300mM, 70mM to 300mM, 80mM to 300mM, 90mM to 300mM, 100mM to 300mM, 150mM to 300mM, 180mM to 300mM, 200mM to 300mM, 2mM to 260mM, 5mM to 260mM, 10mM to 260mM, 20mM to 260mM, 30mM to 260mM, 40mM to 260mM, 50mM~260mM, 60mM~ 12 0mM to 260mM, 150mM to 260mM, 200mM to 260mM, 2mM to 200mM, 5mM to 200mM, 10mM to 200mM, 20mM to 200mM, 30mM to 200mM, 40mM to 200mM, 50mM to 200mM, 60mM to 200mM, 70mM to 200mM, 80mM to 200mM, 90m M~200mM, 2mM~ 150mM, 5mM to 150mM, 10mM to 150mM, 20mM to 150mM, 30mM to 150mM, 40mM to 150mM, 50mM to 150mM, 60mM to 150mM, 70mM to 150mM, 80mM to 150mM, 90mM to 150mM, 2mM to 100mM M, 5mM to 100mM, 10mM to 100mM, 20mM to 100mM, 30mM to 100mM, 40mM to 100mM, 50mM to 100mM, 60mM to 100mM, 70mM to 100mM, 80mM to 100mM, 90mM to 100mM, 2mM to 75mM, 5mM to 75mM, 10mM to 7 5mM, 20mM~ 75mM, 30mM to 75mM, 40mM to 75mM, 50mM to 75mM, 60mM to 75mM, 70mM to 75mM, 2mM to 50mM, 5mM to 50mM, 10mM to 50mM, 20mM to 50mM, 30mM to 50mM, 40mM to 50mM, 2mM to 40mM, It may be present at a concentration of 5mM to 40mM, 10mM to 40mM, 20mM to 40mM, or 30mM to 40mM.

In another embodiment, _MgCl2 may be present at a concentration of 75mM to 300mM.

In another embodiment, _MgCl2 may be present at a concentration of 100mM to 300mM.

In another embodiment, _MgCl2 may be present at a concentration of 200mM to 300mM.

In another embodiment, _MgCl2 may be present at a concentration of 220mM to 260mM.

In another embodiment, _MgCl2 may be present at a concentration between 240mM and 260mM.

In other embodiments, the aqueous compositions or methods according to any embodiment include monovalent salts and divalent salts, preferably at any of the concentrations described above.

In another embodiment, an aqueous composition or method according to any embodiment comprises a monovalent salt at a concentration of 60mM to 80mM and a divalent salt at a concentration of 220 to 260mM.

In another embodiment, an aqueous composition or method according to any embodiment comprises NaCl at a concentration of 40mM to 150mM and _MgCl2 at a concentration of 220mM to 260mM.

In another embodiment, an aqueous composition or method according to any embodiment comprises NaCl at a concentration of 70mM to 150mM and _MgCl2 at a concentration of 220mM to 260mM.

In another embodiment, an aqueous composition or method according to any embodiment comprises NaCl at a concentration of 60mM to 80mM and _MgCl2 at a concentration of 220mM to 260mM.

Further Preferred Embodiments Aqueous compositions or methods according to any embodiment described herein may include one or more amino acid(s). Preferred amino acids are histidine, arginine, lysine-glycine and/or glutamic acid or their salts, especially the L-isomer L-histidine, L-arginine, L-lysine, L-glycine and/or L-glutamic acid or their salts. be. Said amino acid(s) are not amino acids encoded by the recombinant or non-recombinant viruses of the invention. Therefore, this amino acid is not included in the composition through the purification process of the virus (eg, MVA), but is added during the production of the composition to manufacture the vaccine.

The amino acids are preferably present in a concentration of less than 150mM, less than 130mM, less than 120mM, preferably less than 110mM.

In further embodiments, the amino acid is present at a concentration ranging from 10mM to 110mM, 20mM to 110mM, 30mM to 110mM, 40mM to 110mM, 50mM to 110mM, 60mM to 110mM, 70mM to 110mM, or 80mM to 110mM.

In further embodiments, the amino acid is present at a concentration ranging from 40mM to 110mM.

In a further embodiment, the amino acid is present at a concentration ranging from 90mM to 110mM.

In a further embodiment, the amino acid is present at a concentration of 100mM.

In a further embodiment, the amino acid is present at a concentration of 50mM.

In further embodiments, the aqueous compositions of the invention may include histidine (preferably L-histidine) at a concentration of 40mM to 60mM.

In a further embodiment, the aqueous composition of the invention may comprise arginine (preferably L-arginine) at a concentration of 40mM to 60mM.

In a further embodiment, the aqueous composition of the invention may comprise lysine (preferably L-lysine) at a concentration of 40mM to 60mM.

In a further embodiment, the aqueous composition of the invention may comprise glycine (preferably L-glycine) at a concentration of 40mM to 60mM.

In a further embodiment, the aqueous composition of the invention may comprise histidine, arginine, lysine and glycine, preferably each at a concentration of 40mM to 60mM.

In a further embodiment, the aqueous composition of the invention may comprise histidine, arginine, lysine and glycine, preferably each at a concentration of 40mM to 60mM.

The above amino acid concentrations may be used for any of the specific amino acids mentioned in the sections above (eg, histidine, arginine, lysine and/or glycine).

Glutamic acid or a salt thereof (eg, sodium glutamate or sodium glutamate monohydrate) is preferably present at a concentration between 2.5mM and 7.5mM.

In a further embodiment, glutamic acid or a salt thereof (eg, monosodium glutamate monohydrate or monosodium glutamate monohydrate) is present at a concentration ranging from 3mM to 6mM. Preferably, glutamic acid or a salt thereof (eg, monosodium glutamate monohydrate or monosodium glutamate monohydrate) is present at a concentration of 5mM.

Aqueous compositions or methods according to any embodiment described herein may further include octanoate. Preferably, the aqueous composition or method according to any embodiment comprises octanoate ion at a concentration of 5 mM or less, preferably 1 mM or less, more preferably 0.001 to 1 mM.

Aqueous compositions or methods according to any embodiment described herein may include one or more additional carriers, additives, antibiotics, preservatives, adjuvants, and/or diluents, preferably any additional It may further include carriers, additives, antibiotics, preservatives, adjuvants, and/or diluents (pharmaceutically acceptable).

In other embodiments, the aqueous compositions of the invention are vaccines or pharmaceutical compositions.

In further embodiments, the aqueous compositions of the invention are substantially free of HPBCD.

In further embodiments, the aqueous compositions of the invention are free of HPBCD.

In further embodiments, the aqueous compositions of the invention are substantially free of mannitol.

In further embodiments, the aqueous compositions of the invention are free of mannitol.

In further embodiments, the aqueous compositions of the invention are substantially free of citrate.

In further embodiments, the aqueous compositions of the invention are citrate-free.

In further embodiments, the aqueous compositions of the invention are substantially free of chelating agents. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylene glycoltetraacetic acid (EGTA). , dimercaptosuccinic acid (DMSA), diethylenetriaminepentaacetic acid (DTPA), and 2,3-dimercapto-1-propanesulfonic acid (DMPS). "Substantially free of chelating agent" according to the present invention means less than 50 μM of chelating agent.

In further embodiments, the aqueous compositions of the invention are free of chelating agents.

In further embodiments, the aqueous compositions of the invention are substantially free of polysorbate. An example of polysorbate is polysorbate 80.

In further embodiments, the aqueous compositions of the invention are polysorbate-free.

In further embodiments, the aqueous compositions of the invention are substantially free of C ₂ -C ₃ alcohols, where the C ₂ -C ₃ alcohol is ethanol and/or isopropanol. "Substantially free of C ₂ -C ₃ alcohol" according to the present invention means less than 0.05 (v/v) of ethanol or isopropanol.

In further embodiments, the aqueous compositions of the invention are substantially free of C ₂ -C ₃ alcohols, where the C ₂ -C ₃ alcohol is ethanol and/or isopropanol.

In further embodiments, the aqueous compositions of the invention are substantially free of mannitol, citrate, chelating agents, _C2 - _C3 alcohols (ie, ethanol and/or isopropanol), and/or polysorbates.

In further embodiments, the aqueous compositions of the invention are substantially free of mannitol, citrate, chelating agents, _C2 - _C3 alcohols (ie, ethanol and/or isopropanol), and/or polysorbates.

In further embodiments, the aqueous compositions of the invention are substantially free of HPBCD and mannitol.

In further embodiments, the aqueous compositions of the invention are free of HPBCD and mannitol.

In further embodiments, the aqueous compositions of the invention are substantially free of HPBCD, mannitol and citrate.

In further embodiments, the aqueous compositions of the invention are free of HPBCD, mannitol and citrate.

In further embodiments, the aqueous compositions of the present invention are substantially free of HPBCD, mannitol, citrate, chelating agents, and C ₂ -C ₃ alcohols (ie, ethanol and/or isopropanol).

In further embodiments, the aqueous compositions of the invention are free of HPBCD, mannitol, citrate, chelating agents, and _C2 - _C3 alcohols (ie, ethanol and/or isopropanol).

In further embodiments, the aqueous compositions of the invention are substantially free of HPBCD, mannitol, citrate, chelating agents, C2 _{- C3} alcohols (ie, ethanol and/or isopropanol), and polysorbates.

In further embodiments, the aqueous compositions of the invention are substantially free of HPBCD, mannitol, citrate, chelating agents, C2 _{- C3} alcohols (ie, ethanol and/or isopropanol), and polysorbates.

In other embodiments, aqueous compositions according to the invention are contained in vials. The term "vial" refers to any container, container, cartridge, device, glass ampoule, or syringe that can store an active pharmaceutical ingredient, such as a virus as disclosed herein. Accordingly, the terms vial, container, container, cartridge, device, glass ampoule, or syringe may be used interchangeably. Vials are usually made of inert materials, especially glass (such as DIN 2R Type I borosilicate glass virus) or polymeric materials. In a preferred embodiment, the composition is contained in a DIN 2R Type I borosilicate glass vial. In a preferred embodiment, the composition is contained in a syringe.

Compositions of the invention may be administered to a subject, preferably a human, by any means known in the art. Routes of administration include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous administration, intranasal administration, transdermal administration, transcutaneous administration, or percutaneous administration. ) administration. Mode of administration, dosage and frequency of administration can be optimized by those skilled in the art in known manner. In a preferred embodiment, the aqueous compositions of the invention are suitable for parenteral administration or application. In other preferred embodiments, the aqueous compositions of the invention are suitable for intranasal administration or application. In other preferred embodiments, the aqueous compositions of the invention are suitable for intramuscular or subcutaneous administration or application.

In certain embodiments, the aqueous compositions of any embodiment of the invention exhibit at least 50%, 60%, 79% of the starting infectivity (day 0) when stored at +5°C for 3 months. , further defined as having 80% or 90% infectivity.

In certain embodiments, the aqueous compositions of any embodiment of the present invention exhibit at least 70%, 80% or 90% of the starting infectivity (day 0) when stored at -20°C for 3 months. It is further defined as having an infectivity of %.

In certain embodiments, the aqueous compositions of any embodiment of the present invention exhibit at least 70%, 80% or 90% of their starting infectivity (day 0) when stored at +5°C for 6 months. It is further defined as having the infectivity of

In certain embodiments, the aqueous compositions of any embodiment of the invention exhibit at least 70%, 80%, or 90% of the starting infectivity (day 0) when stored at -20°C for 6 months. It is further defined as having an infectivity of %.

In certain embodiments, the aqueous composition of any embodiment of the present invention has at least 70% of its initial infectivity (day 0) when stored at +25°C/60% relative humidity for 5 months; Further defined as having 80% or 90% infectivity.

According to certain embodiments, the aqueous compositions of the present invention are stable.

According to a particular embodiment, the aqueous compositions of the invention have an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0.4 log ₁₀ InfU/mL for at least 3 months at +5°C. mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1); It is stable.

According to a particular embodiment, the aqueous compositions of the invention have an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0.4 log ₁₀ InfU/mL, for at least 6 months at +5°C. mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1); It is stable.

According to a particular embodiment, the aqueous compositions of the invention have an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0.4 log ₁₀ InfU/mL, for at least 9 months at +5°C. mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1); It is stable.

According to a particular embodiment, the aqueous compositions of the invention have an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0.4 log ₁₀ InfU/mL, for at least 12 months at +5°C. mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1); It is stable.

According to a particular embodiment, the aqueous compositions of the invention have an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0.4 log ₁₀ InfU for at least 3 months at -20°C. /mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1). , stable.

According to a particular embodiment, the aqueous compositions of the invention have an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0.4 log ₁₀ InfU for at least 6 months at -20°C. /mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1). , stable.

According to a particular embodiment, the aqueous compositions of the invention have an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0.4 log ₁₀ InfU for at least 9 months at -20°C. /mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1). , stable.

According to a particular embodiment, the aqueous compositions of the invention have an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0.4 log ₁₀ InfU for at least 12 months at -20°C. /mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1). , stable.

According to a particular embodiment, the aqueous composition of the invention has an overall loss of viral titer of less than 0.5 log ₁₀ InfU/mL, preferably 0, for at least 5 months at +25° C./60% relative humidity. less than .4 log ₁₀ InfU/mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably determined by fluorescence-activated cell sorter (FACS) assay according to Example 1) ), it is stable.

According to certain embodiments, the aqueous compositions of the invention are stable for a period of at least 12 months at about -20°C, followed by storage for 3 months at +2 to +8°C, where -20°C During the specified period of storage at −20° C. for at least 12 months, followed by at least 3 months at +2 to +8° C., the overall loss in virus titer is less than 0.5 log ₁₀ InfU/mL. Preferably, the overall loss in viral titer is less than 0.4 log ₁₀ InfU/mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably (as determined by fluorescence-activated cell sorter (FACS) according to Example 1).

According to certain embodiments, the aqueous compositions of the invention are stable for a period of at least 12 months at about -20°C, followed by storage for 9 months at +2 to +8°C, where at least 12 months During storage at −20° C. for the specified period of 1 month and then at +2 to +8° C. for at least 9 months, the overall loss in virus titer is less than 0.5 log ₁₀ InfU/mL. Preferably, the overall loss in viral titer is less than 0.4 log ₁₀ InfU/mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably (as determined by fluorescence-activated cell sorter (FACS) assay according to Example 1).

According to certain embodiments, the aqueous compositions of the invention are stable for a period of at least 24 months at about -20°C, followed by storage at +2 to +8°C for a period of 9 months, where at least 24 months During storage at −20° C. for the specified period of 1 month and then at +2 to +8° C. for at least 9 months, the overall loss in virus titer is less than 0.5 log ₁₀ InfU/mL. Preferably, the overall loss in viral titer is less than 0.4 log ₁₀ InfU/mL, more preferably less than 0.3 log ₁₀ InfU/mL, most preferably less than 0.2 log ₁₀ InfU/mL (preferably (as determined by fluorescence-activated cell sorter (FACS) according to Example 1).

"Overall loss of virus titer" according to the invention refers to the viral titer measured during storage of the composition at the indicated temperature n (e.g. +5°C) and time t (e.g. 6 months). (expressed as log ₁₀ InfU/mL). The overall loss of virus titer is expressed as xlog ₁₀ (eg, as 0.5log ₁₀ for 6 months at +5°C).

According to certain embodiments, the aqueous compositions of the present invention are stable, and when stored at -20°C for a period of 12 months, the compositions exhibit a loss of potency of less than 0.3 log ₁₀ InfU/mL. .

According to certain embodiments, the aqueous compositions of the present invention are stable such that when stored at +4°C to +8°C for a period of 12 months, the compositions exhibit a loss in potency of less than 0.3 log ₁₀ InfU/mL. shows.

According to certain embodiments, the aqueous compositions of the present invention are stable such that when stored at -20° C. for a period of 12 months, the compositions exhibit a loss of potency of less than 0.2 log ₁₀ InfU/mL. .

According to certain embodiments, the aqueous compositions of the present invention are stable such that when stored at +4°C to +8°C for a period of 12 months, the compositions exhibit a loss in potency of less than 0.2 log ₁₀ InfU/mL. shows.

According to certain embodiments, the aqueous compositions of the present invention are stable, and when stored at -20° C. for a period of 12 months, the compositions exhibit a loss of potency of less than 0.1 log ₁₀ InfU/mL. .

According to certain embodiments, the aqueous compositions of the present invention are stable such that when stored at +4°C to +8°C for a period of 12 months, the compositions exhibit a loss in potency of less than 0.1 log ₁₀ InfU/mL. shows.

According to certain other embodiments, the aqueous compositions of the invention are liquid or liquid frozen compositions.

According to certain other embodiments, the aqueous compositions of the invention are aqueous freezing compositions.

According to certain other embodiments, the aqueous compositions of the present invention are aqueous liquid compositions.

According to certain other embodiments, the aqueous compositions of the invention are not dry compositions, and preferably are not freeze-dried or lyophilized compositions.

In another aspect, an aqueous composition of the invention is provided for use as a medicament or vaccine.

Another aspect provides an aqueous composition of the invention for treating or preventing a disease, preferably an infection or cancer.

Another aspect provides the use of an aqueous composition of the invention for the manufacture of a medicament or vaccine for treating or preventing an infectious disease or cancer.

Another aspect provides a method of treating or preventing an infectious disease, comprising administering to a subject a composition of any embodiment of the invention.

The present invention also further provides the following non-limiting embodiments.
1. An aqueous composition comprising at least one poxvirus, at least one disaccharide, sorbitol, gelatin, albumin, a pharmaceutically acceptable buffer, and at least one monovalent salt, such composition comprising: It has a pH in the range of pH 7.0 to pH 8.5.
2. The composition according to embodiment 2, wherein the buffer is a Tris buffer or a phosphate buffer.
The composition of embodiment 2, comprising a Tris buffer at a concentration ranging from 3.1 mM to 50 mM.
The composition of embodiment 2, comprising a Tris buffer at a concentration ranging from 4.1 mM to 15 mM.
A composition according to embodiment 2, comprising a Tris buffer at a concentration ranging from 5.5mM to 11mM.
A composition according to embodiment 2, comprising a Tris buffer at a concentration ranging from 6.7mM to 10mM.
7. The composition according to any one of embodiments 1-6, wherein the disaccharide is trehalose, sucrose, or a combination thereof.
8. The composition according to any one of embodiments 1 to 6, wherein the disaccharide is trehalose.
9. The composition according to any one of embodiments 1 to 6, wherein the disaccharide is sucrose.
10.2% (w/v) to 12% (w/v), 4% (w/v) to 12% (w/v), or 7% (w/v) to 11% (w/v) The composition of any one of embodiments 1 to 9, comprising a concentration of the above disaccharide in the range of .
A composition according to any one of embodiments 1 to 10, comprising a concentration of said disaccharide ranging from 11.7% (w/v) to 11% (w/v).
A composition according to any one of embodiments 1 to 10, comprising a concentration of said disaccharide ranging from 12.7% (w/v) to 11% (w/v).
13.0.2% (w/v) to 5% (w/v), 0.1% (w/v) to 3% (w/v), preferably 1% (w/v) and 3 The composition according to any one of embodiments 1 to 12, comprising sorbitol at a concentration in the range of % (w/v).
A composition according to embodiment 12, comprising sorbitol at a concentration of 14.2% (w/v).
15. The composition according to any one of embodiments 1-14, wherein the composition has a pH in the range of pH 7.3 to pH 8.1.
16. The composition according to any one of embodiments 1-14, wherein the composition has a pH of 7.7.
17.0.1% (w/v) to 5% (w/v), 0.1% (w/v) to 1.2% (w/v), preferably 0.2% (w/v) The composition according to any one of embodiments 1 to 16, comprising albumin at a concentration ranging from v) to 1.2% (w/v).
18. The composition according to any one of embodiments 1 to 17, wherein the gelatin is a gelatin hydrolyzate.
19.0.25% (w/v) ~ 5% (w/v), 0.2% (w/v) ~ 4% (w/v), 0.5% (w/v) ~ 3% (w/v), 0.5% (w/v) to 2.2% (w/v), or 1% (w/v) to 3% (w/v), preferably 2 % (w/v) concentration of gelatin.
20. The composition of Example 1, wherein the composition comprises a disaccharide at a concentration ranging from 4% (w/v) to 12% (w/v), 1% (w/v) to 3% (w/v). sorbitol at a concentration ranging from 0.8% (w/v) to 1.2% (w/v); albumin at a concentration from 1% (w/v) to 3% (w/v); ) and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5 to 25 mM); It has a pH.
21. An aqueous composition comprising at least one poxvirus, wherein the composition comprises a disaccharide selected from trehalose and sucrose at a concentration ranging from 4% (w/v) to 12% (w/v); Sorbitol at a concentration ranging from .5% (w/v) to 3% (w/v), gelatin hydrolyzate at a concentration from 0.2% (w/v) to 3% (w/v), and a medicament. the composition has a pH in the range of 7.3 to 8.1.
22. An aqueous composition comprising at least one poxvirus, wherein the composition comprises trehalose, 0.5% (w/v) at a concentration ranging from 4% (w/v) to 12% (w/v). sorbitol at a concentration ranging from ~3% (w/v), gelatin hydrolyzate at a concentration from 0.2% (w/v) to 3% (w/v), and a pharmaceutically acceptable buffer ( Tris buffer (preferably at a concentration in the range of 5mM to 25mM), said composition has a pH in the range of 7.3 to 8.1.
23. at least one poxvirus, sucrose at a concentration ranging from 4% (w/v) to 12% (w/v), at a concentration ranging from 0.5% (w/v) to 3% (w/v); sorbitol, gelatin hydrolyzate at a concentration of 0.2% (w/v) to 3% (w/v), and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5mM to 25mM). ), said composition having a pH in the range of 7.3 to 8.1.
24. An aqueous composition comprising at least one poxvirus, wherein the composition comprises a disaccharide selected from trehalose and sucrose at a concentration ranging from 4% (w/v) to 12% (w/v); % (w/v) to 3% (w/v) of sorbitol, a concentration of 1% (w/v) to 3% (w/v) of gelatin hydrolyzate, and a pharmaceutically acceptable (preferably Tris buffer at a concentration in the range of 5mM to 25mM), said composition having a pH in the range of 7.3 to 8.1.
25. An aqueous composition comprising at least one poxvirus, the composition comprising trehalose at a concentration ranging from 4% (w/v) to 12% (w/v), 1% (w/v) to 3. % (w/v), gelatin hydrolyzate at a concentration ranging from 1% (w/v) to 3% (w/v), and a pharmaceutically acceptable buffer (preferably from 5mM to Tris buffer at a concentration in the range of 25mM) and the composition has a pH in the range of 7.3-8.1.
26. at least one poxvirus, sucrose at a concentration ranging from 4% (w/v) to 12% (w/v), sorbitol at a concentration ranging from 1% (w/v) to 3% (w/v); an aqueous gelatin hydrolyzate at a concentration of 1% (w/v) to 3% (w/v) and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5mM to 25mM) The composition has a pH in the range of 7.3 to 8.1.
27. An aqueous composition comprising at least one poxvirus, wherein the composition comprises a disaccharide selected from trehalose and sucrose at a concentration ranging from 4% (w/v) to 12% (w/v); Sorbitol at a concentration ranging from .5% (w/v) to 3% (w/v), albumin at a concentration from 0.2% (w/v) to 1.2% (w/v), 0.2 % (w/v) to 3% (w/v) of gelatin hydrolyzate, and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5mM to 25mM), as described above. The composition has a pH ranging from 7.3 to 8.1.
28. An aqueous composition comprising at least one poxvirus, wherein the composition comprises trehalose, 0.5% (w/v) at a concentration ranging from 4% (w/v) to 12% (w/v). Sorbitol at concentrations ranging from ~3% (w/v), albumin at concentrations ranging from 0.2% (w/v) to 1.2% (w/v), 0.2% (w/v) ~3 % (w/v) of gelatin hydrolyzate and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5mM to 25mM); It has a pH in the range of .1.
29. at least one poxvirus, sucrose at a concentration ranging from 4% (w/v) to 12% (w/v), at a concentration ranging from 0.5% (w/v) to 3% (w/v); Sorbitol, albumin at a concentration ranging from 0.2% (w/v) to 1.2% (w/v), gelatin hydrate at a concentration from 0.2% (w/v) to 3% (w/v) an aqueous composition comprising a lysate and a pharmaceutically acceptable buffer (preferably a Tris buffer at a concentration ranging from 5mM to 25mM), the composition comprising: It has a pH.
30. An aqueous composition comprising at least one poxvirus, wherein the composition comprises a disaccharide selected from trehalose and sucrose at a concentration ranging from 4% (w/v) to 12% (w/v); Sorbitol at a concentration ranging from % (w/v) to 3% (w/v), albumin at a concentration ranging from 0.8% (w/v) to 1.2% (w/v), 1% ( gelatin hydrolyzate at a concentration of 3% (w/v) to 3% (w/v), and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5mM to 25mM); has a pH ranging from 7.3 to 8.1.
31. An aqueous composition comprising at least one poxvirus, the composition comprising trehalose at a concentration ranging from 4% (w/v) to 12% (w/v), 1% (w/v) to 3. % (w/v), albumin at a concentration ranging from 0.8% (w/v) to 1.2% (w/v), 1% (w/v) to 3% ( gelatin hydrolyzate at a concentration of 7.3 to 8.1 w/v) and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5mM to 25mM). It has a pH in the range of .
32. at least one poxvirus, sucrose at a concentration ranging from 4% (w/v) to 12% (w/v), sorbitol at a concentration ranging from 1% (w/v) to 3% (w/v); albumin at a concentration of 0.8% (w/v) to 1.2% (w/v), gelatin hydrolyzate at a concentration of 1% (w/v) to 3% (w/v), and pharmaceutical an aqueous composition comprising a buffer (preferably a Tris buffer at a concentration in the range of 5mM to 25mM) that is acceptable to the above, and has a pH in the range of 7.3 to 8.1.
33. An aqueous composition comprising at least one poxvirus, wherein the composition comprises a disaccharide selected from trehalose and sucrose in a concentration in the range of 10% (w/v), in the range of 2% (w/v). sorbitol at a concentration of 1% (w/v), albumin at a concentration of 1% (w/v), gelatin hydrolyzate at a concentration of 3% (w/v), and a pharmaceutically acceptable buffer (preferably in the range of 5mM to 25mM). Tris buffer) and the composition has a pH in the range of 7.3 to 8.1.
34. an aqueous composition comprising at least one poxvirus, said composition comprising: a disaccharide selected from trehalose at a concentration of 10% (w/v); sorbitol at a concentration ranging from 2% (w/v); , albumin at a concentration of 1% (w/v), gelatin hydrolyzate at a concentration of 3% (w/v), and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5mM to 25mM). The composition has a pH in the range of 7.3 to 8.1.
35. An aqueous composition comprising at least one poxvirus, the composition comprising 10% (w/v) sucrose, sorbitol at a concentration in the range of 2% (w/v), 1% (w/v) albumin at a concentration of 3% (w/v), gelatin hydrolyzate at a concentration of 3% (w/v), and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging from 5mM to 25mM); has a pH ranging from 7.3 to 8.1.
36. A composition according to any one of embodiments 1 to 35, wherein the composition comprises sodium chloride or potassium chloride, preferably sodium chloride.
37. The composition of embodiment 36 comprising sodium chloride at a concentration ranging from 50 mM to 150 mM.
38. The composition of embodiment 36, comprising sodium chloride at a concentration ranging from 50 mM to 110 mM.
39. The composition according to any one of embodiments 36-38, further comprising magnesium chloride.
40. The composition of embodiment 39 comprising magnesium chloride at a concentration ranging from 40.1 mM to 300 mM.
41. The composition of embodiment 39 comprising magnesium chloride at a concentration ranging from 100mM to 300mM.
42. The composition of embodiment 39 comprising magnesium chloride at a concentration ranging from 200mM to 300mM.
43. 43. The composition according to any one of embodiments 1-42, further comprising up to four different types of amino acids selected from arginine, serine, histidine, lysine, and glycine.
44. 45. A composition according to embodiment 44, wherein the amino acid is present in the composition at a concentration of less than 120mM, preferably less than 60mM.
45. The composition according to any one of embodiments 1-44, further comprising glutamic acid or a salt thereof.
46. 46. A composition according to embodiment 45, wherein glutamic acid or a salt thereof is present in the composition at a concentration of less than 12mM, preferably less than 6mM.
47. 47. The composition of any one of embodiments 1-46, wherein the composition is suitable for parenteral use.
48. 47. The composition of any one of embodiments 1-46, wherein the composition is suitable for intranasal use.
49. A composition according to any one of embodiments 1 to 46, wherein the composition is suitable for intramuscular or subcutaneous use.
50. The composition of any one of embodiments 1-49, wherein the composition is a liquid composition or a liquid frozen composition.
51. The composition of any one of embodiments 1-49, wherein the composition is an aqueous freezing composition.
52. 52. The composition of any one of embodiments 1-51, wherein the poxvirus is vaccinia virus.
53. 53. The composition of any one of embodiments 1-52, wherein the poxvirus is modified vaccinia Ankara virus (MVA).
54. The composition according to any one of embodiments 1-53, wherein the virus is a recombinant virus.
55. 55. A composition according to embodiment 54, wherein said recombinant virus comprises a nucleic acid expressing an antigen, preferably a viral antigen.
56. 56. The composition of embodiment 54 or 55, wherein the recombinant virus comprises a nucleic acid encoding a respiratory syncytial virus (RSV) antigen.
57. 57. The composition of any one of embodiments 54-56, wherein the recombinant virus comprises a nucleic acid encoding a respiratory syncytial virus (RSV) protein.
58. 58. The composition of any one of embodiments 54-57, wherein the recombinant virus comprises a nucleic acid encoding RSV F glycoprotein.
59. 59. The composition of any one of embodiments 54-58, wherein the recombinant virus comprises nucleic acids encoding RSV F glycoprotein, two RSV G glycoproteins, RSV N protein, and RSVM2-1 protein.
60. 60. The composition of any one of embodiments 1-59, wherein the virus titer in the composition is between 1×10 ⁶ InfU/mL and 1×10 ¹⁰ InfU/mL.
61. 61. The composition of any one of embodiments 1-60, wherein the composition is substantially citrate-free.
62. 62. The composition of any one of embodiments 1-61, wherein the composition is substantially free of mannitol.
63. 61. The composition of any one of embodiments 1-60, wherein the composition is citrate-free.
64. 62. The composition of any one of embodiments 1-61, wherein the composition is free of mannitol.
65. Embodiments, wherein the composition exhibits a loss of potency of less than 0.3 (preferably 0.2, most preferably 0.1) log ₁₀ InfU/mL when stored at +4°C to +8°C for a period of 12 months. 65. The composition according to any one of items 1 to 64.
66. A vaccine or pharmaceutical composition comprising a composition of an embodiment according to any one of embodiments 1-65.
67. A composition according to any one of embodiments 1 to 65 for use as a medicament or vaccine.
68. A composition according to any one of embodiments 1 to 65 for treating or preventing a disease, preferably an infectious disease.
69. Use of a composition according to any one of embodiments 1 to 65 of the invention for the manufacture of a medicament or vaccine for treating or preventing an infectious disease.
70. A method of treating or preventing an infectious disease, comprising administering to a subject a therapeutically effective amount of a composition according to any one of embodiments 1-65.
71. A method of making an aqueous live poxvirus composition comprising the following steps:
a. providing a preparation comprising at least one live poxvirus in a pharmaceutically acceptable buffer;
b. combining the poxvirus preparation of step a) with a solution comprising a disaccharide, sorbitol, gelatin, albumin, and a pharmaceutically acceptable buffer, and at least one monovalent salt;
The above method, wherein the buffers of a) and b) have a pH in the range of pH 7.0 to pH 8.5.
72. 72. The method of embodiment 71, wherein the buffer is a Tris buffer or a phosphate buffer.
73. 73. The method of embodiment 72, wherein the Tris buffer has a concentration ranging from 1 mM to 25 mM.
74. 74. The method of any one of embodiments 72 or 73, wherein the Tris buffer has a concentration in the range of 1mM to 15mM.
75. 74. The method of any one of embodiments 72-73, wherein the Tris buffer has a concentration in the range of 5mM to 11mM.
76. 74. The method of any one of embodiments 72-73, wherein the Tris buffer has a concentration in the range of 7mM to 10mM.
77. 77. The method of any one of embodiments 71-76, wherein the buffers of a) and b) have a pH in the range of 7.3 to 8.1.
78. 78. The method of any one of embodiments 71-77, wherein the disaccharide is trehalose, sucrose, or a combination thereof.
79. 78. The method of any one of embodiments 71-77, wherein the disaccharide is trehalose.
80. 78. The method of any one of embodiments 71-77, wherein the disaccharide is sucrose.
81. Any one of embodiments 71-80, wherein after combining a) and b), said combined composition comprises a disaccharide in a concentration ranging from 4% (w/v) to 12% (w/v). The method described in section.
82. 81. The method of any one of embodiments 71-80, wherein after combining a) and b), the combined composition comprises a disaccharide at a concentration of 10% (w/v).
83. Any one of embodiments 71-82, wherein after combining a) and b), the combined composition comprises sorbitol at a concentration ranging from 1% (w/v) to 3% (w/v). The method described in.
84. 83. The method of any one of embodiments 71-82, wherein after combining a) and b), the combined composition comprises sorbitol at a concentration of 2% (w/v).
85. After combining a) and b), the combined composition has a concentration ranging from 0.2% (w/v) to 3% (w/v), preferably from 1% (w/v). 85. The method of any one of embodiments 71-84, comprising gelatin at a concentration in the range of 3% (w/v).
86. After combining a) and b), the combined composition has a concentration ranging from 0.2% (w/v) to 3% (w/v), preferably from 1% (w/v). 86. The method according to any one of embodiments 71-85, comprising gelatin (preferably gelatin hydrolyzate) at a concentration in the range of 3% (w/v).
87. After combining a) and b), the combined composition has a concentration in the range of 0.2% (w/v) to 3% (w/v), preferably 0.2% (w/v). ) to 1.2% (w/v).
88. 72. The method of embodiment 71, wherein after combining a) and b), the combined composition has a concentration ranging from 2% (w/v) to 12% (w/v). disaccharide (preferably trehalose, sucrose or a combination thereof), sorbitol at a concentration ranging from 0.5% (w/v) to 3% (w/v), 0.2% (w/v) to Gelatin hydrolyzate at a concentration of 3% (w/v), albumin at a concentration ranging from 0.2% (w/v) to 1.2% (w/v), and a pharmaceutically acceptable buffer. (preferably a Tris buffer at a concentration in the range of 5-25mM), wherein the composition has a pH in the range of 7.3-8.1.
89. Embodiments 71-88, wherein said monovalent salt is sodium chloride or potassium chloride, preferably after combining a) and b), the combined composition comprises sodium chloride at a concentration ranging from 50mM to 150mM. The method according to any one of the above.
90. 90. A method according to embodiment 89, further comprising magnesium chloride, preferably after combining a) and b), the combined composition comprising magnesium chloride at a concentration ranging from 1 mM to 300 mM.
91. 90. A method according to embodiment 89, further comprising magnesium chloride, preferably after combining a) and b), the combined composition comprising magnesium chloride at a concentration in the range of 200mM to 300mM.
92. 92. The method of any one of embodiments 71-91, wherein the poxvirus is vaccinia virus.
93. 92. The method of any one of embodiments 71-91, wherein the poxvirus is a modified vaccinia Ankara (MVA) virus.
94. 94. The method of any one of embodiments 71-93, wherein the virus is a recombinant virus.
95. 95. A method according to embodiment 94, wherein said recombinant virus comprises a nucleic acid expressing an antigen, preferably a viral antigen.
96. 95. The composition of embodiment 94, wherein the recombinant virus comprises a nucleic acid encoding a respiratory syncytial virus (RSV) antigen.
97. 95. The composition of embodiment 94, wherein the recombinant virus comprises a nucleic acid encoding a respiratory syncytial virus (RSV) protein.
98. 98. The method of any one of embodiments 96 or 97, wherein the recombinant virus comprises a nucleic acid encoding RSV F glycoprotein.
99. 99. The method of embodiment 98, wherein the recombinant virus comprises nucleic acids encoding RSV F glycoprotein, two RSV G glycoproteins, RSV N protein, and RSVM2-1 protein.

Definitions and Terminology It is to be understood that both the foregoing summary and detailed description are exemplary and explanatory only and are not limiting of the claimed invention. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the claims appended hereto. I also want to be understood. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Terms are defined and explained so that the invention may be more easily understood. Further definitions are provided throughout the detailed description.

Note that as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "nucleic acids" include one or more nucleic acid sequences, and references to "methods" include modifications to or substitutions of the methods described herein. Contains reference to possible equivalent steps and methods known to those skilled in the art.

Unless stated otherwise, the term "at least" preceding a series of elements should be understood to refer to every element of the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

As used herein, the conjunction "and/or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and/or," the first option refers to the applicability of the first element in the absence of the second element. The second option refers to the applicability of the second element in the absence of the first element. The third option indicates that the first and second elements can be applied together. Any one of these options is understood to be within the meaning and therefore meets the requirements of the term "and/or" as used herein. The simultaneous applicability of multiple options is also understood to be within the meaning and therefore meets the requirements of the term "and/or".

Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are used in reference to is meant to include an integer or step, or group of integers or steps, but not exclude any other integer or step, or group of integers or steps. When used in the context of aspects or embodiments in the description of the invention, the term "comprising" may be modified and therefore replaced by the term "comprising" or "comprising" or may be replaced by the term "having" as used herein. Similarly, any of the foregoing terms (comprising, containing, including, having) whenever used in the context of aspects or embodiments in the description of the invention By virtue of this, we include the terms "consisting of" or "consisting essentially of," each of which has a specific legal meaning depending on the jurisdiction.

As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the essential novel features of the claims.

As used herein, the term "substantially free" of ingredients means, unless otherwise specified herein, trace amounts of ingredients that do not substantially affect the stability of the compositions of the present invention. not excluded. For example, the term "free of" before mannitol means that the aqueous composition of the present invention is free of mannitol.

"About" as used in this application means ±10% unless otherwise specified. It is also noted that, unless stated otherwise, any numerical value, such as a concentration or concentration range, mentioned herein should be understood to be modified by the term "about" in all cases. . Throughout this specification, the term "about" in reference to any amount or concentration is considered to include that amount. For example, "about 5mM" is intended herein to include 5mM, as well as values understood to be about 5mM with respect to the described entity. As used herein, the use of numerical ranges includes all possible subranges within such ranges, including whole numbers and fractional parts of values, unless the context clearly dictates otherwise. Explicitly include all individual numbers in . Similarly, the term "about" preceding any numerical value or range used herein in the context of the present invention may be omitted, less preferably without the term "about," and this May be replaced with a number or range.

The terms "nucleic acid," "nucleotide sequence," "nucleic acid sequence," and "polynucleotide" may be used interchangeably and may be linear or branched, single-stranded or double-stranded, or a hybrid thereof. Refers to RNA or DNA. Polynucleotides may be obtained by chemical synthesis or may be derived from microorganisms. The term "exogenous" nucleic acid sequence when used in connection with a recombinant virus refers to a foreign nucleic acid sequence, a nucleic acid sequence not contained in the non-recombinant virus used to generate the recombinant virus, or a recombinant virus. Refers to a nucleic acid sequence that is inserted into the viral genome during production.

"Pharmaceutically acceptable" means that the dosage and concentration of carriers, excipients, antibiotics, preservatives, adjuvants, diluents, stabilizers, or excipients used are compatible with the subject to whom they are administered ( means not causing substantially any undesirable or harmful effect(s) on the subject(s). A "pharmaceutically acceptable" excipient is any inert substance that is combined with an active molecule, such as a virus, to prepare a preferred or convenient dosage form. A "pharmaceutically acceptable" excipient is one that is non-toxic to recipients at the dosages and concentrations used and is compatible with other components of the formulation, including the viral preparation. Examples of excipients are antifreezes, nonionic surfactants, buffers, salts, and inhibitors of free radical oxidation. A "pharmaceutically acceptable carrier" is described, for example, in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed. , Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, eds. , Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed. , Pharmaceutical Press (2000).

"Stable", "stabilized", "stability" or "stabilizing", all of which can be used interchangeably, means that the poxvirus contained in the composition of the invention is essentially the physical stability, identity, integrity, and/or chemical stability, identity, integrity, particle form, and/or biological activity or potency of the pharmaceutical composition as necessary for its shelf life; It is understood that it is to be held when As used herein, the term "shelf-life" refers to the period under specified storage conditions (e.g., storage at +2°C to +8°C) for human pharmaceutical use. means the time during which a product remains active and/or stable according to its product characteristics. Retention period corresponds to the point at which a specified lower or upper limit of specifications is exceeded.

Stability refers to various characteristics such as the amount of poxvirus (e.g., MVA) in the formulation (the amount of poxvirus in the formulation), potency, and/or other quality aspects over a period of time and under a particular storage condition. can be evaluated by determining the These characteristics of poxvirus (eg, MVA) preparations can be measured at elevated temperatures (predicting real-time temperature) or other stress conditions. For example, formulations may be subjected to +20°C incubation, +25°C, -20°C or +5°C, or to freeze/thaw cycles and agitation to study the effectiveness of different formulations to maximize shelf life. You may. Methods for determining the stability of poxviruses (e.g. MVA) are well known to those skilled in the art and include at least one method selected from the group of visual inspection, pH measurements, turbidity assays, particle morphology and potency (infectivity) assays. can be determined by two methods.

Turbidity measurements are the intensity of transmitted light due to scattering of particles within a sample detected at wavelengths at which molecules within the sample do not absorb the light (e.g. 350 nm for samples where proteins are the predominant chromophore). Measure the loss (apparent absorbance). When molecules aggregate or form supramolecular complexes, the light scattering that was random when coming from separate particles now becomes coherent, thereby increasing the measured intensity. This makes light scattering and turbidity measurements useful techniques for detecting aggregation and complex formation or dissociation.

For turbidity assays, samples are transferred in triplicate to UV-transparent flat-bottom microplates. Absorbance spectra are recorded by a microplate reader from 230 to 500 nm, and absorbance at 975 nm is measured to determine and optionally correct for path length differences. A control sample consisting of a formulation without MVA was included in the assay to correct for scattering or absorption of matrix components if necessary. Apparent absorbance at 350 nm was used as a quantitative measure of turbidity.

Turbidity assay indicates stability of MVA samples. Aggregation of MVA increases turbidity and reduces capsid dissociation. The accuracy of the assay is <5% (CV%) with turbidity values >1 NTU.

Methods of determining particle morphology are well known to those skilled in the art. For example, particle morphologies are described in, for example, Schweneker et al. It may be determined using transmission electron microscopy and immunoelectron microscopy (immuno-EM), as described in J Virol 2017, 91:e00343-00317. Alternative methods for determining particle morphology are described, for example, by Filipe et al. Nanoparticle Tracking Analysis (NTA) described in Pharm Res 2010, 27:796-810. Nanoparticle tracking analysis (NTA) is a method for directly and real-time visualization and analysis of particle size distribution and aggregation in liquids. Based on laser illumination microscopy technology, the Brownian motion of nanoparticles is analyzed in real time by a charge-coupled device (CCD) camera, and each particle is simultaneously but separately visualized and tracked by a dedicated particle tracking image analysis program. Ru. The ability of NTA to simultaneously measure particle size and particle scattering intensity allows for the separation of heterogeneous particle mixtures and direct estimation of particle concentration.

When used in connection with viruses as used herein, the term "potency" or "infectivity" refers to the ability of a virus to infect a cell, referring to the entry and multiplication of the virus in the cell or organism. ing. Infectivity therefore refers to the activity of a poxvirus (eg, vaccinia virus or MVA) expressed as infectious units (InfU), usually given as InfU/mL. Both the terms "potency" and "infectivity" may be used interchangeably in the present invention. The efficacy of poxviruses such as MVA is determined using various methods known to those skilled in the art, for example, determining the percentage of virus-positive cells such as baby hamster kidney cells 21 (BHK-21) after viral infection. You may. A preferred assay is, for example, a fluorescence activated cell sorter (FACS) assay as described in the Examples.

The terms "subject" and "patient" are used interchangeably. As used herein, a subject is typically a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). , and in some preferred embodiments human.

According to the invention, "virus" means viruses, virus particles and viral vectors. All of these terms may be used interchangeably. The term includes wild-type virus, recombinant and non-recombinant virus, live virus, and live attenuated virus.

According to the invention, concentrations given in % (w/v) mean weight in grams (g) per volume in 100 mL, for example 20% (w/v) means 20 g/100 mL. means. Concentrations expressed in % (v/v) mean weight in mL per 100 mL volume, for example 20% (v/v) means 20 mL/100 mL.

Throughout the specification, unless otherwise stated, values for physical parameters such as pH are those measured at +25°C.

Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Incorporated. To the extent that material incorporated by reference contradicts or is inconsistent with this specification, this specification will supersede such material. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The practice of the present invention will employ, unless otherwise specified, conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant technology, all within the skill of the art. be. For example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology. , Ausubel FM, et al. , eds, 1987; the series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR , eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988 checking ...

The following examples are illustrative of the invention and should not be construed as limiting the scope of the claims in any way. They merely serve to clarify the invention.

Example 1: Potency (Infectivity) Assay The titer (InfU/mL) of recombinant or non-recombinant MVA was determined by fluorescence activated cell sorter (FACS) assay. MVA-infected Baby Hamster Kidney Cell 21 (BHK-21) cells were immunostained with a fluorochrome-conjugated antibody specific for vaccinia virus (VACV) and then analyzed for quantitative cell counting. Acquired and quantified using a FACSVerse™ (BD Bioscience) instrument equipped with a BD flow sensor.

More specifically, 2.5×10 ⁵ BHK 21 cells (source ATCC) were seeded in GMEM/9% FBS/1.8% Ala-Gln in 12-well plates. The next day, cells were infected with serial dilutions of the desired MVA virus stock. After incubation for 1 hour at 37 degrees, rifampin (100 μg/mL/9% FBS/1.8% Ala-Gln in GMEM) was added. Cells were harvested 19±2 hours after infection, fixed and permeabilized with the BD Perm/Wash™ kit before antibody staining. Fixed cells were incubated with anti-vaccinia FITC (Fitzgerald Industries International, catalog number 60-v68) for 60-90 minutes. The percentage of virus-positive cells was then determined by flow cytometry using a BDFACSVerse™ cytometer. Total cell numbers were determined using a BDFACSVerse™ flow sensor with unstained cells fixed in parallel. Calculation of virus titer (InfU/mL) was based on the percentage of virus-positive cells, the virus dilution used during infection, the infection volume, and the average number of cells per well. To limit the effect of well on well variation in cell number, cell number was established by averaging the cell number of multiple wells. Only dilutions containing 2-35% VACV-positive cells were included in the calculation of InfU/mL of virus samples. InfU per mL of sample dilution (InfU/mL) was calculated according to the following formula.

Example 2: Production of Recombinant MVA The recombinant MVA used for the stability studies in the example was MVA-BN RSV (MVA-mBN294B), which is described in WO2015/136056, which is incorporated herein by reference. there were. MVA-mBN294B encodes the RSV-F (Along) protein, G (A) and G (B) proteins, and N and M2-1 proteins, where the N and M2-1 sequences are the 2A self of the FMDV virus. Encoded by a single open reading frame separated by truncated protease domains. IGR64/65 and IGR148/149 were used as antigen integration sites. This gene was expressed under the poxvirus promoter Pr7.5e/l (Cochran et al. J Virol 1985, 54:30-37), PrS (Chakrabarti et al. BiotoChnIQUES 1997, 23: 1094-1097), PRLE1 (BAUR ET AL.J VIROL 2010, 84: 8743-8752) and PRH5M (Wennier et al.Plos ONE 2013, 8: E73511) 。

MVA-BN RSV (MVA-mBN294B) was produced in chicken embryonic fibroblast (CEF) cells. CEF cells were produced using standard methods well known to those skilled in the art as described in WO2012/010280. Several methods are available to amplify recombinant MVA virus and purify bulk drug substance that are also well known to those skilled in the art (eg, as described in WO2006/052826). One exemplary method used is described below.

MVA-BN RSV (MVA-mBN294B) was cultured using 50L wave bag culture in VP-SFM medium supplemented with 4mM L-glutamine, 0.01% pluronic and antibiotics (e.g. gentamicin 100μg/mL). Produced in CEF cells. Cells were harvested and homogenized by sonication to lyse the cells and release the generated virus particles. After centrifugation, depth filtration and benzonase treatment, the virus preparation was further purified and concentrated by ultrafiltration and diafiltration, finally in 10mM Tris, 140mM NaCl, pH 7.7±0.4. Purified bulk drug substance was obtained. BDS was stored at -80°C ± 10°C.

Example 3: Vaccine formulation A blend of two MVA-BN RSV batches was used as starting material (BDS). The two batches were concentrated to a final titer of 9.129 Log ₁₀ (InfU/mL), corresponding to 1.35 x 10 ⁹ InfU/mL, at pH 7.7 ± 0 containing 10 mM Tris and 140 mM NaCl. .4 diafiltration buffer. Residual host cell DNA was 46 μg/mL.

Such concentrated BDS of MVA-BN RSV was made into 2x concentrated formulation buffer (final 1x concentration 10mM Tris, 70mM NaCl) containing various stabilizers, which was filtered using a 0.2μm syringe before use. Mixed. The sample was filled into a DIN2-R glass vial (0.25 mL). Details of the liquid formulation and the ingredients used are shown in Table 1. The following raw materials were used for the preparation of the 2x formulation buffer: NEOSORB® PF pyrogen-free sorbitol, GMP, Roquette, FR; sucrose, Sigma Aldrich; trehalose, GMP, Pfanstiehl GmbH; chloride Magnesium hexahydrate, Ph. Eur. Sigma; glycine, Pharmagrade, USP, Sigma Aldrich; L-glutamic acid, monosodium salt, PharmaGrage (MSG), Sigma; recombinant human albumin, Recombumin®, Albumedix, hydrolyzed gelatin, Vaccipro® ), Gelita; L-Arginine, PharmaGrade, USP, Sigma Aldrich; L-Histidine, PharmaGrade, USP, Sigma Aldrich; Dextran 70, USP, Pharmacosmos; Sodium Chloride BioXtra Sig ma; Tris hydrochloride, PharmaGrade, Sigma Aldrich. MVA-mBN294B in 10mM Tris and 140mM sodium chloride (pH 7.7±0.4) was used as a control.

For each formulation, samples were used three times at +5 ± 3°C (0, 3, 5, 6, 7, 8, and 12 months) or -20°C (0, 4, 6 months, and 12 months). aliquoted for storage and analysis at various times. At each time point, vials were stored under the respective storage conditions of the corresponding MVA-BN RSV formulation for potency analysis using fluorescence-activated cell sorter (FACS) assay as a stability indicator test according to Example 1. Fill with 0.25 mL of each. The pH and appearance were also tested.

Example 4: Linear regression analysis -+5 and -20°C
The stability slopes of the formulations were analyzed by linear regression in descending order (i.e., the lowest absolute slope value/ The most stable ones are listed first).

Summary of Results Real-time stability data shows that aqueous compositions of F23, F9, and F17, when stored at refrigerator temperature (i.e., +5°C), compared to prior art compositions (control) and dextran-based compositions such as F27. It is shown that it showed improved stability than the formulation. Gelatin hydrolysate and albumin, sorbitol compositions added to disaccharides, especially trehalose or sucrose, give the best stability results and limited virus loss for at least 12 months when stored between +5°C. (Table 1). This allows the supply of unfrozen vaccine products at +2°C to +8°C. Further formulation development studies have shown that the stability of aqueous MVA-mBN294B can be further improved by storing it frozen at -20°C for at least 12 months after drug manufacture, and thawing to a maximum temperature of +2°C to +8°C. It was shown that it provided sufficient storage when stored for a limited time of 9 months. This was not expected based on the results described by Prabhu et al., who used a lyophilized camelpox vaccine containing 3.5% hydrolysed gelatin and 3.5% sorbitol after reconstitution. The formulation showed an immediate drop in virus titer at 4° C., with a loss of 2 log ₁₀ TCID ₅₀ of virus within 10 days (Prabhu et al. Biologicals 2014, 42:169-175).

Example 5: Linear Regression Analysis - +25°C/60% Relative Humidity The virus formulations specified in Table 3 were exposed to +25±2°C at 60%±5% relative humidity. At the beginning of the study, samples corresponding to time point zero were transferred to -80°C. After 5 weeks (35 days) of incubation, samples of each formulation were obtained for determination of virus titer.

Incubation of the formulation continued for a total of 10 weeks. To assess the pH stability of the virus preparations, pH was measured at the beginning of the study and after 10 weeks.

Viral titers were determined using a FACS assay as described in Example 1. Samples were analyzed in parallel (same assay). The reduction in virus titer was measured in triplicate and each replicate pair was analyzed on a different day.

The stability slopes of the formulations incubated for 5 weeks (weeks) at +25 ± 2 °C and 60% ± 5% relative humidity (RH) were determined by linear regression analysis and are listed in Table 3 in descending order (i.e., the lowest absolute slope value /restabilization first). Table 3 also shows the decrease in titer log ₁₀ (InfU/mL) and the decrease in pH.

Summary of Results As described in Example 4, formulation F23 demonstrated improved stability of MVA-BN-RSV when stored at +5°C or -20°C for 12 months compared to the control. Indicated. Furthermore, the stability of formulation F23 was improved over the control even after 26 months at -20°C (data not shown).

Improved stability was also observed when MVA-BN-RSV formulated in F23 was stored at +25 ± 2 °C/60% ± 5% relative humidity for 5 weeks (see Table 3, Figure 3). thing). Similarly, when stored for 10 weeks at +25°C/60% relative humidity, the pH of F23 was more stable than the control formulation (see Table 3).

More specifically, compared to the other formulations, formulation F23 showed the best results in terms of titer reduction and slope (Table 3, Figure 3), as well as pH reduction (Table 3).

Formulations F66, F55, and F62 contained trehalose, while F23 contained sucrose instead. However, this difference is considered irrelevant with respect to virus stability.

Additionally, F23 differed from other formulations in that it contained gelatin. Comparing F23 and F66, it was clear that gelatin and albumin provided better stability than albumin alone.

Therefore, gelatin and albumin added to compositions containing sorbitol and disaccharides (e.g. sucrose) provide the best stability results when stored at +25°C and 60% relative humidity, with no virus loss over at least 5 weeks. was limited. This allows shipping and storage of unfrozen vaccine products under conditions of high ambient temperature and humidity, such as in regions where warm, humid climates predominate.

Although many aspects of the invention herein are illustrated or illustrated with MVA-BN, particularly MVA-mBN294B, the principles embodied by the invention apply to other poxvirus strains as well as other poxvirus strains including them. It is also applicable to recombinant poxvirus strains and compositions and should not necessarily be limited to the particular strains/viruses used herein. Accordingly, other recombinant and non-recombinant poxviruses and compositions containing them are also within the scope of the invention.

It will be apparent that the precise details of the methods or compositions described herein may be changed or modified without departing from the spirit of the invention as described. We claim all such changes and variations that come within the scope and spirit of the following claims.
Note that this application relates to the invention described in the claims, but may also include the following as other aspects.
1. An aqueous composition comprising at least one poxvirus, at least one disaccharide, sorbitol, gelatin, albumin, a pharmaceutically acceptable buffer, and at least one monovalent salt, the composition having a pH of 7.0 to 8.5. The composition has a pH in the range of .
2. 3. The composition according to 2 above, wherein the buffer is a Tris buffer or a phosphate buffer.
3. The composition according to claim 2, comprising said Tris buffer at a concentration ranging between 3.1 and 50 mM.
4. 4. The composition according to any one of 1 to 3 above, wherein the disaccharide is trehalose, sucrose, or a combination thereof.
5. A composition according to any one of 1 to 4 above, comprising a concentration of said disaccharide ranging between 5.2% (w/v) and 12% (w/v).
6. A composition according to any one of claims 1 to 5, comprising sorbitol at a concentration ranging between 0.2% (w/v) and 5% (w/v).
7. 7. According to any one of 1 to 6 above, the gelatin is a gelatin hydrolyzate, preferably at a concentration ranging between 0.25% (w/v) and 5% (w/v). Composition.
8. A composition according to any one of claims 1 to 7, comprising albumin at a concentration ranging between 0.1% (w/v) and 5% (w/v).
9. 1. The composition according to claim 1, wherein the composition comprises 1% (w/v) and Sorbitol at a concentration ranging between 3% (w/v), albumin at a concentration ranging between 0.2% (w/v) and 1.2% (w/v), 0.5% ( gelatin hydrolyzate at a concentration between 3% (w/v) and 3% (w/v), and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration ranging between 5 and 25 mM). , wherein said composition has a pH in the range between 7.3 and 8.1.
10. Composition according to any one of claims 1 to 9, wherein said composition comprises sodium chloride or potassium chloride, preferably sodium chloride.
11. A method of making an aqueous live poxvirus composition comprising the following steps:
a. providing a preparation comprising at least one live poxvirus in a pharmaceutically acceptable buffer;
b. combining the poxvirus preparation of step a) with a solution comprising at least one disaccharide, sorbitol, gelatin, albumin, and a pharmaceutically acceptable buffer, and at least one monovalent salt; including,
The method, wherein the buffers of a) and b) have a pH in the range between pH 7.0 and pH 8.5.
12. 12. The method according to 11 above, wherein the buffer is a Tris buffer or a phosphate buffer.
13. 12. The method according to claim 11, wherein after combining a) and b), said combined composition is in a range between 2% (w/v) and 12% (w/v). disaccharide (preferably trehalose, sucrose or a combination thereof) at a concentration of 0.2% (preferably trehalose, sucrose or a combination thereof), sorbitol at a concentration ranging between 0.5% (w/v) and 3% (w/v); gelatin hydrolyzate at a concentration between 0.2% (w/v) and 1.2% (w/v); albumin, and a pharmaceutically acceptable buffer (preferably Tris buffer at a concentration between 5 and 25 mM), wherein said composition has a concentration between 7.3 and 8.1. The above method, having a pH of
14. The composition according to any one of paragraphs 1 to 10 or the method according to any one of paragraphs 11 to 13, wherein said poxvirus is a vaccinia virus, preferably a modified vaccinia Ankara (MVA) virus.
15. The composition according to any one of 1 to 10 or 14 above, wherein said virus is a recombinant virus, preferably said recombinant virus comprises a nucleic acid encoding a respiratory syncytial virus (RSV) antigen, or The method according to any one of 11 to 14 above.

References Al Yaghchi, C. , Zhang, Z. , Alusi, G. , Lemoine,N. R. , and Wang, Y. (2015) Vaccinia virus, a promising new therapeutic agent for pancreatic cancer. Immunotherapy 7:1249-1258.
Baur, K. , Brinkmann, K. , Schweneker, M. , Patzold, J. , Meisinger-Henschel, C. , Hermann, J. , Steigerwald,R. , Chaplin, P. , Suter, M. , and Hausmann, J. (2010). Immediate-early expression of a recombinant antigen by modified vaccinia virus ankara breaks the immunodominance of strong vector-specific B8R antigen in acute and memory CD8 T-cell responses. J Virol 84:8743-8752.
Berhanu, A. , King, D. S. , Mosier, S. , Jordan, R. , Jones, K. F. , Hruby, D. E. , and Grosenbach, D. W. (2010). Impact of ST-246(R) on ACAM2000 smallpox vaccine reactogenicity, immunogenicity, and protective efficacy in immunodeficiency mi ce. Vaccine 29:289-303.
Burke, C. J. , Hsu, T. A. , and Volkin, D. B. (1999). Formulation, stability, and delivery of live attenuated vaccines for human use. Crit Rev Ther Drug Carrier Syst 16:1-83.
Capelle, M. A. H. , Babich, L. , van Deventer-Troost, J. P. E. , Salerno,D. , Krijgsman, K. , Dirmeier, U. , Raaby, B. , and Adriansen, J. (2018). Stability and suitability for storage and distribution of Ad26. ZEBOV/MVA-BN(R)-Filo heterologous prime-boost Ebola vaccine. Eur J Pharm Biopharm 129:215-221.
Chakrabarti, S. , Sisler, J. R. , and Moss, B. (1997). Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 23:1094-1097.
Choi, Y. , and Chang, J. (2013). Viral vectors for vaccine applications. Clin Exp Vaccine Res 2:97-105.
Cochran, M. A. , Puckett, C. , and Moss, B. (1985). In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals ．． J Virol 54:30-37.
Filipe, V. , Hawe, A. , and Jiskoot, W. (2010). Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregation regates. Pharm Res 27:796-810.
Hekker, A. C. , J. M. B. , and Smith, L. (1973). A stable frozen-dried smallpox vaccine made in monolayer cultures of primary rabbit kidney cells. Journal of Biological Standardization 1:21-32.
Hodge, J. W. , Sabzevari, H. , Yafal, A. G. , Gritz, L. , Lorenz, M. G. , and Schlom, J. (1999). A triad of costimulation molecules synergize to amplify T-cell activation. Cancer Res 59:5800-5807.
Just, I. , and Finke, H. (1979). [A contribution to the stabilization of the MVA-vaccine (author's transl)]. Zentralbl Bakteriol Orig A 245:276-282.
Leon, A. , David, A. L. , Madeline, B. , Guianvarc'h, L. , Dureau, E. , Champion-Arnaud, P. , Hebben,M. , Huss, T. , Chatrenet, B. , and Schwamborn, K.; (2016). The EB66(R) cell line as a valuable cell substrate for MVA-based vaccines production. Vaccine 34:5878-5885.
Mastrangelo, M. J. , Eisenlohr, L. C. , Gomella, L. , and Lattime, E. C. (2000). Poxvirus vectors: orphaned and underappreciated. J Clin Invest 105:1031-1034.
Mayr, A. , and Danner, K. (1978). Vaccination against pox diseases under immunosuppressive conditions. Dev Biol Stand 41:225-234.
Mayr, A. , , Hochstein-Mintzel, V. , and Stickl, H. (1975). Abstammung, Eigenschaften und Verwendung des attenuierten Vaccinia-Stammes MVA. Infection 3:6-14
Meyer, H. , Sutter, G. , and Mayr, A. (1991). Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their infection on virus. J Gen Virol 72(Pt 5):1031-1038.
Otagiri, M. , and Chuang, V. T. (2009). Pharmaceutically important pre- and posttranslational modifications on human serum albumin. Biol Pharm Bull 32:527-534.
Prabhu, M. ,Bhanuprakash,V. , Venkatesan, G. , Yogisharadhya, R. , Bora, D. P. , and Balamurugan, V. (2014). Evaluation of stability of live attenuated camelpox vaccine stabilized with different stabilizers and reconstituted wit h various diluents. Biologicals 42:169-175.
Rezaee, F. , Linfield, D. T. ,,Harford,T. J. , and Piedimonte, G. (2017). Ongoing developments in RSV prophylaxis: a clinician's analysis. Curr Opin Virol 24:70-78.
Schweneker, M. , Laimbacher, A. S. , Zimmer, G. , Wagner, S. , Schraner, E. M. , Wolferstatter, M. , Klingenberg, M. , Dirmeier, U. , Steigerwald, R. , Lauterbach, H. , et al. (2017). Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles. J Virol 91:e00343-00317.
Suter, M. , Meisinger-Henschel, C. , Tzatzaris, M. , Hulsemann, V. , Lukassen, S. , Wulff, N. H. , Hausmann, J. , Howley, P. , and Chaplin, P. (2009). Modified vaccinia Ankara strains with identificational coding sequences actually represent complex mixtures of viruses that dete rmine the biological properties of each strain. Vaccine 27:7442-7450.
Tlaxca, J. L. , Ellis, S. , and Remmele, R. L. , Jr. (2015). Live attenuated and inactivated viral vaccine formulation and nasal delivery: potential and challenges. Adv Drug Deliv Rev 93:56-78.
Vanderplasschen, A. , and Pastoret, P. P. (2003). The uses of poxviruses as vectors. Curr Gene Ther 3:583-595.
Verheust, C. , Goossens, M. , Pauwels, K. , and Breyer, D. (2012). Biosafety aspects of modified vaccinia virus Ankara (MVA)-based vectors used for gene therapy or vaccination. Vaccine 30:2623-2632.
Wennier, S. T. , Brinkmann, K. , Steinhausser, C. , Maylander, N. , Mnich, C. , Wielert, U. , Dirmeier, U. , Hausmann, J. , Chaplin, P. , and Steigerwald, R. (2013). A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses. PLoS One 8:e73511.
Zhang, Y. -z. , Jiang, C. -l. , Yu, X. -h. , Lou, C. -p. , Zhao, D. -h. , Wu, Y. -g. , Jin, Y. -h. , Liu, C. -s. , and Kong,W. (2007). Immunogenicity of Lyophilized MVA Vaccine for HIV-1 in Mice Model. Chemical Research in Chinese Universities 23:329-332.

Claims (21)

An aqueous composition comprising at least one poxvirus, at least one disaccharide, sorbitol, gelatin, albumin, a pharmaceutically acceptable buffer, and at least one monovalent salt, the composition having a pH of 7.0 to 8.5. wherein the disaccharide is trehalose, sucrose, or a combination thereof, and the poxvirus is vaccinia virus . 2. The composition of claim 1 , wherein the buffer is a Tris buffer or a phosphate buffer. 3. The composition of claim 2, comprising the Tris buffer at a concentration ranging between 1 and 50 mM. Composition according to any one of claims 1 to 3 , comprising a concentration of said disaccharide ranging between 2% (w/v) and 12% (w/v). Composition according to any one of claims 1 to 4 , comprising sorbitol at a concentration ranging between 0.2% (w/v) and 5% (w/v). The composition according to any one of claims 1 to 5 , wherein the gelatin is a gelatin hydrolyzate. Composition according to any one of claims 1 to 5, wherein the gelatin is a gelatin hydrolyzate with a concentration ranging between 0.25% (w/v) and 5% (w/v). thing. Composition according to any one of claims 1 to 7, comprising albumin at a concentration ranging between 0.1% (w/v) and 5% (w/v). 2. The composition of claim 1, wherein the composition comprises 1% (w/v) disaccharide at a concentration ranging between 4% (w/v) and 12% (w/v). and 3% (w/v) sorbitol, and albumin in a concentration range between 0.2% (w/v) and 1.2% (w/v), 0.5%. (w/v) and 3% (w/v) of gelatin hydrolyzate, and a pharmaceutically acceptable buffer, said composition having a concentration of between 7.3 and 8.1 The composition has a pH in the range of . 2. The composition of claim 1, wherein the composition comprises 1% (w/v) disaccharide at a concentration ranging between 4% (w/v) and 12% (w/v). and 3% (w/v) sorbitol, and albumin in a concentration range between 0.2% (w/v) and 1.2% (w/v), 0.5%. (w/v) and 3% (w/v) of gelatin hydrolyzate, and a concentration of Tris buffer ranging between 5 and 25 mM, said composition comprising 7.3 and 3% (w/v) of Tris buffer. 8.1. A composition according to any one of claims 1 to 10 , wherein the composition comprises sodium chloride or potassium chloride. A method of making an aqueous live poxvirus composition comprising the following steps:
a. providing a preparation comprising at least one live poxvirus in a pharmaceutically acceptable buffer;
b. combining the poxvirus preparation of step a) with a solution comprising at least one disaccharide, sorbitol, gelatin, albumin, and a pharmaceutically acceptable buffer, and at least one monovalent salt; including,
wherein said buffers of a) and b) have a pH in the range between pH 7.0 and pH 8.5, said disaccharide is trehalose, sucrose, or a combination thereof, and said poxvirus The above method , wherein is vaccinia virus . 13. The method of claim 12 , wherein the buffer is a Tris buffer or a phosphate buffer. 13. The method of claim 12 , wherein after combining a) and b), the combined composition contains between 2% (w/v) and 12% (w/v). disaccharide in a range of concentrations, sorbitol in a range of concentrations between 0.5% (w/v) and 3% (w/v), 0.2% (w/v) and 3% (w/v); ), albumin at a concentration ranging between 0.2% (w/v) and 1.2% (w/v), and a pharmaceutically acceptable buffer. wherein said composition has a pH in the range between 7.3 and 8.1. 13. The method of claim 12, wherein after combining a) and b), the combined composition contains between 2% (w/v) and 12% (w/v). disaccharide in a range of concentrations, sorbitol in a range of concentrations between 0.5% (w/v) and 3% (w/v), 0.2% (w/v) and 3% (w/v); ), gelatin hydrolyzate at a concentration between 0.2% (w/v) and 1.2% (w/v), and albumin at a concentration between 5mM and 25mM. wherein said composition has a pH ranging between 7.3 and 8.1. A composition according to any one of claims 1 to 11 , wherein the vaccinia virus is a modified vaccinia Ankara (MVA) virus. 16. The method according to any one of claims 12 to 15, wherein the vaccinia virus is a modified vaccinia Ankara (MVA) virus. The composition according to any one of claims 1 to 11 or 16 , wherein the virus is a recombinant virus . 19. The composition of claim 18, wherein the recombinant virus comprises a nucleic acid encoding a respiratory syncytial virus (RSV) antigen. The method according to any one of claims 12 to 15, wherein the virus is a recombinant virus. 21. The method of claim 20, wherein the recombinant virus comprises a nucleic acid encoding a respiratory syncytial virus (RSV) antigen.